Serum amyloid p derivatives and their preparation and use

ABSTRACT

One aspect of the present invention relates to the surprising discovery that modification of a glycan structure on a human SAP polypeptide can increase the biological activity of the SAP polypeptide relative to a corresponding sample of wild-type SAP isolated from human serum. The disclosure provides both variant human SAP polypeptides and methods for making the same. In particular, the present invention provides methods and compositions for in vitro and in vivo addition, deletion, or modification of sugar residues to produce SAP polypeptides, such as a human SAP polypeptide, having a desired glycosylation pattern.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/941,515, filed Jul. 28, 2020 (pending), which is a continuation ofU.S. patent application Ser. No. 16/731,320, filed Dec. 31, 2019,(abandoned), which is a continuation of U.S. application Ser. No.16/417,537, filed May 20, 2019, (abandoned), which is a continuation ofU.S. patent application Ser. No. 16/156,978, filed Oct. 10, 2018,(abandoned) which is a continuation of U.S. patent application Ser. No.15/492,085, filed Apr. 20, 2017, (abandoned) which is a continuation ofU.S. patent application Ser. No. 15/264,707, filed Sep. 14, 2016(abandoned), which is a continuation of U.S. patent application Ser. No.15/054,640, filed Feb. 26, 2016 (abandoned), which is a continuation ofU.S. patent application Ser. No. 12/794,132, filed Jun. 4, 2010, nowU.S. Pat. No. 9,296,800, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/217,931 (expired), filed on Jun. 4, 2009. All ofthe teachings of each of the above-referenced applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Serum Amyloid P (SAP) is a member of the pentraxin family of proteins.SAP is secreted by the liver and circulates in the blood as a stablepentamer. Previous research demonstrates SAP has an important role inboth the initiation and resolution phases of the immune response. SAPcan bind to sugar residues on the surface of bacteria and therebypromote their opsonization and engulfment by antigen-presenting cells.SAP also binds to free DNA and chromatin generated by apoptotic cells atthe resolution of an immune response, thus preventing a secondaryinflammatory response against these antigens. Molecules bound by SAP areremoved from extracellular areas due to the ability of SAP to bind toall three classical Fcγ. receptors (FcγR), having a particular affinityfor FcγRII (CD32) and FcγRIII (CD16). After receptor binding, SAP andany attached complex are generally internalized and processed by thecell.

Recently, it has been suggested that SAP can be used as a therapeuticagent to treat various disorders, including fibrosis-related disorders,hypersensitivity disorders, autoimmune disorders, mucositis, andinflammatory disorders such as those cause by microbial infection. See,for example, U.S. patent application Ser. Nos. 11/707,333, 12/217,617,12/720,845, and 12/720,847. Protein therapeutics for treating humandisease have revolutionized the health care industry. However, there aremany difficulties in producing a protein therapeutic having thenecessary potency and/or in sufficient quantity to be useful as atherapeutic agent. Many potential therapeutic agents are modified toincrease their biological activity, such as plasma half-life, relativeto the naturally-derived protein. Recombinant expression technology isusually implemented to produce polypeptides in sufficient quantity.Unfortunately, many recombinant systems produce polypeptides havingdifferent biological properties than the naturally-derived forms, whichmay affect the pharmacokinetics, safety, and efficacy of a therapeuticproduct.

Therefore, a need remains for developing SAP polypeptides, and methodsof manufacturing them, suitable for therapeutic treatment of humans.

SUMMARY OF THE INVENTION

In part, the disclosure provides variant Serum Amyloid P (SAP)polypeptides and methods for producing them. The present inventionincludes methods and compositions for in vitro and in vivo addition,deletion, or modification of sugar residues to produce variant SAPpolypeptides having a desired glycosylation pattern.

In certain aspects, the disclosure provides a glycosylated human SAPpolypeptide, comprising an N-linked or O-linked oligosaccharide chainthat has at least one branch terminating with a α2,3-linked sialic acidmoiety.

In certain aspects, the disclosure provides a glycosylated human SAPpolypeptide, comprising an N-linked or O-linked oligosaccharide chainthat has at least 50% fewer α2,6-linked sialic acid moieties thanwild-type SAP isolated from human serum.

In certain aspects, the disclosure provides methods of making aglycosylated human SAP polypeptide, comprising expressing a SAPpolypeptide in a cell and isolating the SAP polypeptide from the cell.In a preferred embodiment, the cell is a CHO cell. In certain aspects,the cell is a CHO-S cell.

In certain aspects, the disclosure provides methods of making a humanSAP polypeptide, comprising expressing a human SAP polypeptide in a CHOcell and isolating the human SAP polypeptide from the cell.

In certain aspects, the disclosure provides methods of making a humanSAP polypeptide, comprising providing a glycosylated human SAPpolypeptide containing an N-linked or O-linked oligosaccharide chain andenzymatically or chemically altering the N-linked or O-linkedoligosaccharide chain of the SAP polypeptide to produce a modifiedglycosylated SAP polypeptide.

In certain aspects, the disclosure provides methods of making a humanSAP polypeptide, comprising providing a human SAP polypeptide andenzymatically or chemically altering the SAP polypeptide to produce aglycosylated SAP polypeptide comprising an N-linked or O-linkedoligosaccharide.

In certain aspects, the disclosure provides a human SAP polypeptideprepared by a process comprising expressing a SAP polypeptide in a CHOcell and isolating the SAP polypeptide from the cell.

In certain aspects, the disclosure provides a CHO cell that contains ahuman SAP polypeptide with an N-linked oligosaccharide chain having atleast one branch of the oligosaccharide chain terminating with aα2,3-linked sialic acid moiety.

In certain aspects, the disclosure provides a CHO cell containing apolynucleotide sequence encoding a human SAP polypeptide.

In certain aspects, the disclosure provides a human SAP polypeptidehaving an IC50 for inhibiting the differentiation of monocytes intofibrocytes in vitro that is less than one-half, less than one-third,less than one-fourth, less than one-tenth, or less than one-hundredththan that of a corresponding sample of wild-type SAP isolated from humanserum.

In preferred embodiments, human SAP polypeptides of the invention havean N-linked oligosaccharide chain. In some embodiments, at least onebranch of the N-linked oligosaccharide chain terminates with aα2,3-linked sialic acid moiety. In some embodiments, the N-linkedoligosaccharide chain has at least 50% fewer α2,6-linked sialic acidmoieties than a wild-type SAP isolated from human serum. In someembodiments, all branches of the N-linked oligosaccharide chainterminate with α2,3-linked sialic acid moieties. In some embodiments,the N-linked oligosaccharide chain is substantially free of α2,6-linkedsialic acid moieties. Glycovariant SAP polypeptides of the invention maycomprise one or more branches, e.g., the N-linked oligosaccharide chainmay be characterized as having a bi-antennary, tri-antennary,tetra-antennary, or a penta-antennary structure. In some embodiments,the N-linked oligosaccharide chain comprises a pentasaccharide core ofMan[(α1,6-)-(Man(α1,3)]-Man(β1,4)-GlcNAc(β1,4)-GlcNAc(β1,N)-Asn. In someembodiments, the N-linked oligosaccharide chain comprises at least onebranch having the structure NeuNAc2α3Galβ4GlcNAcβ2Manα6. In someembodiments, at least one branch of the N-linked oligosaccharide chainis substantially free of galactose and N-acetylglucosamine. In someembodiments, all the branches of the N-linked oligosaccharide chain aresubstantially free of galactose and N-acetylglucosamine. In someembodiments, at least one branch of the N-linked oligosaccharide chaincomprises one or more mannose residues. In some embodiments, theN-linked oligosaccharide chain comprises at least one fucose residue.Any of the glycovariant SAP polypeptides of the invention may compriseat least one modified glycosyl residue. A modified glycosyl residue maybe conjugated to one or more modifying groups selected fromwater-soluble and -insoluble polymers, therapeutic moieties, diagnosticagents, and biomolecules.

In certain aspects, the SAP polypeptide of the invention may be arecombinant polypeptide. SAP polypeptide of the invention may comprisean amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or100% identical to SEQ ID Nos. 1, 2, 3, or 4. Preferably, the SAPpolypeptide is a human SAP protein. A human SAP polypeptide of theinvention may comprise an amino acid sequence at least 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1.

In certain aspects, the human SAP polypeptide of the invention is afusion protein comprising an SAP domain and one or more heterologousdomains. The heterologous domain may enhance one or more of in vivostability, in vivo half-life, uptake/administration, tissue localizationor distribution, formation of protein complexes, and/or purification.

In certain aspects, the human SAP polypeptide of the invention comprisesone or more modified amino acid residues, e.g., a PEGylated amino acid,a glycosylated (e.g., O-linked glycosylation) amino acid, a prenylatedamino acid, an acetylated amino acid, a biotinylated amino acid, and/oran amino acid conjugated to an organic derivatizing agent. The modifiedamino acid residues may enhance one or more of in vivo stability, invivo half-life, uptake/administration, tissue localization ordistribution, formation of protein complexes, and/or purification.

In preferred embodiments, human SAP polypeptides of the invention haveincreased biological activity relative to a corresponding sample ofwild-type SAP isolated from human serum. In certain aspects, the SAPpolypeptides of the invention have an IC₅₀ for inhibiting thedifferentiation of monocytes into fibrocytes in vitro that is less thanone-half, less than one-third, less than one-fourth, less thanone-tenth, or less than one-hundredth that of a corresponding sample ofwild-type SAP isolated from human serum.

In certain aspects, methods of making any of the human SAP polypeptidesof the invention comprise an additional step of enzymatically orchemically altering the SAP polypeptide to attach an N-linked orO-linked oligosaccharide chain to the SAP polypeptide or to modify theexisting N-linked or O-linked oligosaccharide chain of the SAPpolypeptide. In some embodiments, enzymatically or chemically alteringthe SAP polypeptide comprises treating the SAP polypeptide with one ormore enzymatic proteins selected from glycosyltransferases,glycosidases, and phosphatases. In some embodiments, the process ofenzymatically or chemically altering the SAP polypeptide is effected inthe presence of one or more sugar precursors. Suitable sugar precursorsinclude, but are not limited to, UDP-N-acetylglucosamine,CMP-N-glycolylneuraminic acid UDP-N-acetylgalactosamine,CMP-N-acetylneuraminic acid, UDP-galactose, and GDP-fucose. In someembodiments, the process of enzymatically or chemically altering the SAPpolypeptide removes one or more terminal α2,6-linked sialic acidmoieties from the N-linked or O-linked oligosaccharide chain. In someembodiments, the process of enzymatically or chemically altering theisolated SAP polypeptide replaces one or more terminal α2,6-linkedsialic acid moieties on the oligosaccharide chain with one or moreα2,3-linked sialic acid moieties.

The disclosure further provides pharmaceutical preparations of human SAPpolypeptides of the invention suitable for use in a mammal.Pharmaceutical preparations of the invention include at least one of theSAP polypeptides disclosed herein and a pharmaceutically acceptablecarrier. In some embodiments, the pharmaceutical preparation furthercomprises an additional active agent. In some embodiments, thepharmaceutical preparation is prepared as a sustained releaseformulation. In some embodiments, pharmaceutical preparations of thedisclosure are suitable for administration to a patient topically, byinjection, by intravenous injection, by inhalation, by continuous depot,or by pump.

The disclosure further provides methods for treating or preventingSAP-responsive disorders or conditions by administering to a patient inneed thereof a therapeutically effective amount of one or more of theSAP polypeptides of the invention. SAP-responsive disorders orconditions include, but are not limited to, fibrotic orfibroproliferative disorders or conditions, hypersensitivity disordersor conditions, autoimmune disorders or conditions, inflammatory diseasesor conditions, and mucositis. The SAP polypeptide of the invention maybe administered to a patient topically, by injection, by intravenousinjection, by inhalation, by continuous depot or pump, or a combinationthereof In some embodiments, the SAP polypeptide of the invention isadministered with one or more additional active agents.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 . Fibrocyte differentiation assay. An ELISA-based assay was usedto measure production of MDC after incubation of monocytes with SAPpolypeptides. The Y-axis indicates the average potency (i.e., average of7 independent experiments) of human serum-derived SAP (hSAP) compared torecombinant human SAP (rhSAP) isolated from CHO-S cells. Relativeactivity of hSAP is set at 1.0.

FIGS. 2A-2F. Glycan structural analysis of variant SAP polypeptides.Liquid Chromatography Mass Spectrometry (LCMS) analysis (A) andAnion-Exchange High Performance Liquid Chromatography (AEX-HPLC)analysis (B) was used to determine the sialic acid linkages onglycoremodeled recombinant human SAP isolated from CHO-S cells. LiquidChromatography Mass Spectrometry (LCMS) analysis (C) and Anion-ExchangeHigh Performance Liquid Chromatography (AEX-HPLC) analysis (D) was usedto determine the sialic acid linkages on glycoremodeled hSAP (humanserum-derived SAP). Liquid Chromatography Mass Spectrometry (LCMS)analysis (E) and Anion-Exchange High Performance Liquid Chromatography(AEX-HPLC) analysis (F) was used to determine the sialic acid linkageson rhSAP that was treated with an α2,3-sialyltransferase to increase thenumber of terminal 2,3-linked sialic acids on the SAP glycans. For LCMSfigures, the X-axis represents mass in Daltons, and the Y-axis isrepresents relative intensity. For the HPLC traces, the X-axis is thetime in minutes, and the Y-axis is absorbance units (mAU).

FIG. 3 . Fibrocyte differentiation assay. An ELISA-based assay was usedto measure production of MDC after incubation of monocytes with SAPvariant polypeptides. The Y-axis indicates the average relative activityof each SAP variant compared to a hSAP reference standard, for which theactivity is set at 1.0 (see the left-most bar).

FIG. 4 . Fibrocyte differentiation assay. Monocytes were treated withhMCSF and then subsequently quantified for fibrocyte differentiation.The X-axis represents the concentration of hMCSF incubated with donormonocytes. The Y-axis indicates the amount of fibrocyte proliferation atday five as measured by the enumeration of fibrocytes per 5.0×10⁴ cells.

FIGS. 5 . Fibrocyte differentiation assay. Monocytes were treated withhSAP and then subsequently quantified for fibrocyte differentiation. TheX-axis indicates the concentration of hSAP incubated with donormonocytes. The Y-axis indicates the amount of fibrocyte proliferation atday five as measured by the enumeration of fibrocytes per 5.0×10⁴ cells.

DETAILED DESCRIPTION OF THE INVENTION Overview

Most naturally occurring peptides have carbohydrate moieties (i.e.,glycans) attached to the peptide via specific linkages to certain aminoacids along the length of the primary peptide chain, thus forming“glycopeptides.” The glycosylation pattern on any given peptide can haveenormous implications for the function of that peptide. For example, thestructure of the N-linked glycans on a peptide can impact variouscharacteristics of the peptide, including protease susceptibility,intracellular trafficking, secretion, tissue targeting, biologicalhalf-life, and antigenicity. The alteration of one or more of thesecharacteristics greatly affects the efficacy of a peptide in its naturalsetting.

The glycan structures found in naturally occurring glycopeptides aretypically divided into two classes, N-linked and O-linked glycans.Peptides expressed in eukaryotic cells are typically N-glycosylated onasparagine residues at sites in the peptide primary structure containingthe sequence asparagine-X-serine/threonine, where X can be any aminoacid except proline and aspartic acid. The carbohydrate portion of suchpeptides is known as an N-linked glycan or N-linked oligosaccharide. Theearly events of N-glycosylation occur in the endoplasmic reticulum (ER)and are conserved in mammals, plants, insects and other highereukaryotes. First, an oligosaccharide chain comprising fourteen sugarresidues is constructed on a lipid carrier molecule. As the nascentpeptide is translated and translocated into the ER, the entireoligosaccharide chain is transferred to the amide group of theasparagine residue in a reaction catalyzed by a membrane-boundglycosyltransferase enzyme. The N-linked glycan is further processedboth in the ER and in the Golgi apparatus. The further processinggenerally entails removal of some of the sugar residues and addition ofother sugar residues in reactions catalyzed by glycosylases andglycosyltransferases specific for the sugar residues removed and added.

Typically, the final structures of the N-linked glycans are dependentupon the organism in which the peptide is produced. For example,peptides produced in bacteria are generally unglycosylated. Peptidesexpressed in insect cells typically contain high mannose orpauci-mannose N-linked oligosaccharide chains. Peptides produced inmammalian cell culture are usually differentially glycosylated dependingupon the species and cell culture conditions. Even in the same speciesand under the same conditions, a certain amount of heterogeneity in theglycosyl chain is sometimes encountered. In general, peptides producedin plant cells comprise glycan structures that differ significantly fromthose produced in animal cells.

A variety of methods have been proposed in the art to customize theglycosylation pattern of a peptide, including methods described in thePublished International Applications Nos. WO 99/22764, WO 98/58964, andWO 99/54342 as well as in U.S. Pat. No. 5,047,335. Essentially, many ofthe enzymes required for the in vitro glycosylation of peptides havebeen cloned and sequenced. In some instances, these enzymes have beenused in vitro to add specific sugars to a glycan on a peptide. In otherinstances, cells have been genetically engineered to express acombination of enzymes and desired peptides such that addition of adesired sugar moiety to an expressed peptide occurs within the cell.

Two principal classes of enzymes are used in the synthesis ofcarbohydrates: glycosyltransferases and glycosidases.Glycosyltransferases add or modify the existing oligosaccharidestructures on a peptide. Glycosyltransferases are effective forproducing specific products with good stereochemical and regiochemicalcontrol. Glycosyltransferases have been used to prepare oligosaccharidesand to modify terminal N- and O-linked carbohydrate structures,particularly on peptides produced in mammalian cells. For example, theterminal oligosaccharides of glycopeptides can be completely sialylatedand/or fucosylated to provide more consistent sugar structures usingglycosyltransferases, which may improves glycopeptide pharmacodynamicsand a variety of other biological properties.

The glycosidases are further classified as exoglycosidases (e.g.,β-mannosidase, β-glucosidase), and endoglycosidases (e.g. Endo-A,Endo-M). Glycosidases normally catalyze the hydrolysis of a glycosidicbond. However, under appropriate conditions, they can be used to formthis linkage. Most glycosidases used for carbohydrate synthesis areexoglycosidases; the glycosyl transfer occurs at the non-reducingterminus of the substrate. The glycosidase binds a glycosyl donor in aglycosyl-enzyme intermediate that is either intercepted by water toyield the hydrolysis product, or by an acceptor, to generate a newglycoside or oligosaccharide. An exemplary pathway using anexoglycosidase is the synthesis of the core trisaccharide of allN-linked glycopeptides, including the β-mannoside linkage, which isformed by the action of β-mannosidase (Singh et al., Chem. Commun.993-994 (1996)). Although their use is less common than that of theexoglycosidases, endoglycosidases are also utilized to preparecarbohydrates. Endoglycosidases can be used to transfer an entireoligosaccharide chain, rather than a monosaccharide, onto a polypeptide.Oligosaccharide fragments have been added to substrates usingendo-β-N-acetylglucosamines, such as endo-F andendo-M (Wang et al.,Tetrahedron Lett. 37: 1975-1978; and Haneda et al., Carbohydr. Res. 292:61-70 (1996). Each of these classes of enzymes has been successfullyused produce glycosylated peptides. For a general review, see, Crout etal., Curr. Opin. Chem. Biol. 2: 98-111 (1998).

Serum amyloid P (SAP) is a naturally occurring serum protein in mammalscomposed of five identical subunits, or “promoters”, which arenon-covalently associated in a disk-like complex. SAP belongs to thepentraxin super family of proteins, which are characterized by thiscyclic pentameric structure. The classical short pentraxins include SAPas well as C-reactive protein (Osmand, A. P., et al., Proc. Nat. Acad.Sci., 74: 739-743, 1997). SAP is normally synthesized in the liver andhas a physiological half-life of twenty-four hours. The sequence of thehuman SAP subunit is disclosed below, which corresponds to amino acids20-223 of Gene bank Accession NO. NP_001630 (signal sequence notdepicted).

(SEQ ID NO: 1) HTDLSGKVFVFPRESVTDHVNLITPLEKPLQNFTLCFRAYSDLSRAYSLFSYNTQGRDNELLVYKERVGEYSLYIGRHKVTSKVIEKFPAPVHICVSWESSSGIAEFWINGTPLVKKGLRQGYFVEAQPKIVLGQEQDSYGGKFDRSQSFVGEIGDLYMWDSVLPPENILSAYQGTPLPANILDWQALNYEIR GYVIIKPLVWVThe sequence of the Gallus gallus SAP subunit is disclosed below.

(SEQ ID NO: 2) QEDLYRKVFVFREDPSDAYVLLQVQLERPLLNFTVCLRSYTDLTRPHSLFSYATKAQDNEILLFKPKPGEYRFYVGGKYVTFRVPENRGEWEHVCASWESGSGIAEFWLNGRPWPRKGLQKGYEVGNEAVVMLGQEQDAYGGGFDVYNSFTGEMADVHLWDAGLSPDKMRSAYLALRLPPAPLAWG RLRYEAKGDVVVKPRLREALGAThe sequence of the Bos taurus SAP subunit is disclosed below.

(SEQ ID NO: 3) QTDLRGKVFVFPRESSTDHVTLITKLEKPLKNLTLCLRAYSDLSRGYSLFSYNIHSKDNELLVFKNGIGEYSLYIGKTKVTVRATEKFPSPVHICTSWESSTGIAEFWINGKPLVKRGLKQGYAVGAHPKIVLGQEQDSYGGGFDKNQSFMGEIGDLYMWDSVLSPEEILLVYQGSSSISPTILDWQALKYEIK GYVIVKPMVWGThe sequence of the Cricetulus migratorius SAP subunit is disclosedbelow.

(SEQ ID NO: 4) QTDLTGKVFVFPRESESDYVKLIPRLEKPLENFTLCFRTYTDLSRPHSLFSYNTKNKDNELLIYKERMGEYGLYIENVGAIVRGVEEFASPVHFCTSWESSSGIADFWVNGIPWVKKGLKKGYTVKTQPSIILGQEQDNYGGGFDKSQSFVGEMGDLNMWDSVLTPEEIKSVYEGSWLEPNILDWRALNYEMS GYAVIRPRVWH

One aspect of the present invention relates to the surprising discoverythat modification of a glycan structure on a human SAP polypeptide canincrease the biological activity of the SAP polypeptide relative to acorresponding sample of wild-type SAP isolated from human serum. Asdemonstrated by the examples of the disclosure, isolated SAP from humanserum contains only α2,6-linked sialic acid residues. In contrast,recombinant human SAP produced in CHO cells contains only α2,3-linkedsialic acid residues. Using in vitro cell-based bioassays, α2,3-linkedsialic acid SAP polypeptides were demonstrated to have consistentlyhigher activity than wild-type SAP (i.e., SAP comprising α2,6-linkedsialic acid moieties) isolated from human serum. The variant SAPpolypeptides of the invention would be more effective as therapeuticagents due to their increased biological potency. For example, morepotent SAP variants may require lower dosing and/or less frequent dosingrelative to wild-type SAP isolated from human serum. The presentdisclosure provides both variant human SAP polypeptides and methods formaking the same. In particular, the disclosure includes methods andcompositions for in vitro and in vivo addition, deletion, ormodification of sugar residues to produce a human SAP polypeptide havinga desired glycosylation pattern.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art. Generally, the nomenclature used herein andthe laboratory procedures in cell culture, molecular genetics, organicchemistry, and nucleic acid chemistry and hybridization are those wellknown and commonly employed in the art. Standard techniques are used fornucleic acid and peptide synthesis. The techniques and procedures aregenerally performed according to conventional methods in the art andvarious general references (e.g., Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, 2d ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.), which are provided throughout thisdocument.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the terms “treatment” and “treating”, refer to obtaininga desired pharmacologic and/or physiologic effect. The effect may beprophylactic in terms of completely or partially preventing a disorderor symptom thereof and/or may be therapeutic in terms of a partial orcomplete cure for a disorder and/or adverse affect attributable to thedisorder. “Treatment”, as used herein, covers any treatment of a diseasein a mammal, particularly in a human, and includes: (a) increasingsurvival time; (b) decreasing the risk of death due to the disease; (c)inhibiting the disease, i.e., arresting its development or reducing therate of disease progression; and (d) relieving the disease, i.e.,causing regression of the disease.

As used herein, a therapeutic that “inhibits” or “prevents” a disorderor condition is a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample.

As used herein the terms “subject” and “patient” refer to animalsincluding mammals, such as humans. The term “mammal” includes primates,domesticated animals including dogs, cats, sheep, cattle, horses, goats,pigs, mice, rats, rabbits, guinea pigs, captive animals such as zooanimals, and wild animals.

As used herein the term “tissue” refers to an organ or set ofspecialized cells such as skin tissue, lung tissue, kidney tissue, andother types of cells.

The term “therapeutic effect” is art-recognized and refers to a local orsystemic effect in animals, particularly mammals, and more particularlyhumans caused by a pharmacologically active substance. The phrase“therapeutically effective amount” means that amount of such a substancethat produces some desired local or systemic effect at a reasonablebenefit/risk ratio applicable to any treatment. The therapeuticallyeffective amount of such substance will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration,which can readily be determined by one of ordinary skill in the art. Forexample, certain compositions described herein may be administered in asufficient amount to produce a desired effect at a reasonablebenefit/risk ratio applicable to such treatment.

As used herein, the term “nucleic acid” refers to a polynucleotide suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single-stranded (such assense or antisense) and double-stranded polynucleotide.

The terms “peptides”, “proteins” and “polypeptides” are usedinterchangeably herein. The term “purified protein” refers to apreparation of a protein or proteins that are preferably isolated from,or otherwise substantially free of, other proteins normally associatedwith the protein(s) in a cell or cell lysate. The term “substantiallyfree of other cellular proteins” or “substantially free of othercontaminating proteins” is defined as encompassing individualpreparations of each of the proteins comprising less than 20% (by dryweight) contaminating protein, and preferably comprises less than 5%contaminating protein. Functional forms of each of the proteins can beprepared as purified preparations by using a cloned gene as is wellknown in the art. By “purified”, it is meant that the indicated moleculeis present in the substantial absence of other biologicalmacromolecules, such as other proteins (particularly other proteinswhich may substantially mask, diminish, confuse or alter thecharacteristics of the component proteins either as purifiedpreparations or in their function in the subject reconstituted mixture).The term “purified” as used herein preferably means at least 80% by dryweight, more preferably in the range of 85% by weight, more preferably95-99% by weight, and most preferably at least 99.8% by weight, ofbiological macromolecules of the same type present (but water, buffers,and other small molecules, especially molecules having a molecularweight of less than 5000, can be present). The term “pure” as usedherein preferably has the same numerical limits as “purified”immediately above.

“N-linked” oligosaccharides are those oligosaccharides that are linkedto a peptide backbone through asparagine, by way of anasparagine-N-acetylglucosamine linkage. N-linked oligosaccharides arealso called “N-glycans.” Naturally occurring N-linked oligosaccharideshave a common pentasaccharide core ofMan[(α1,6-)-(Man(a1,3)]-Man(β1,4)-GlcNAc(β1,4)-GlcNAc(β1,N). They differin the presence of, and in the number of branches (also called antennae)of peripheral sugars such as N-acetylglucosamine, galactose,N-acetylgalactosamine, fucose, and sialic acid. Optionally, thisstructure may also contain a core fucose molecule and/or a xylosemolecule.

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetyl-neuraminic acid (often abbreviated as Neu5Ac, NeuAc, or NANA).A second member of the family is N-glycolyl-neuraminic acid (Neu5Gc orNeuGc), in which the N-acetyl group of NeuAc is hydroxylated. A thirdsialic acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN)(Nadano et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al.,J. Biol. Chem. 265: 21811-21819 (1990)). Also included are 9-substitutedsialic acids such as a 9-O-C₁C₆-acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac.For review of the sialic acid family, see, e.g., Varki, Glycobiology 2:25-40 (1992); Sialic Acids: Chemistry, Metabolism and Function, R.Schauer, Ed. (Springer-Verlag, New York (1992)).

A “genetically engineered” or “recombinant” cell is a cell having one ormore modifications to the genetic material of the cell. Suchmodifications include, but are not limited to, insertions of geneticmaterial, deletions of genetic material and insertion of geneticmaterial that is extrachromosomal whether such material is stablymaintained or not.

As used herein, the term “modified sugar,” refers to a naturally- ornon-naturally-occurring carbohydrate that is enzymatically added onto anamino acid or a glycosyl residue of a peptide in a process of theinvention. The modified sugar is selected from a number of enzymesubstrates including, but not limited to, sugar nucleotides (mono-, di-,and tri-phosphates), activated sugars (e.g., glycosyl halides, glycosylmesylates) and sugars that are neither activated nor nucleotides. A“modified sugar” maybe covalently functionalized with a “modifyinggroup.” Useful modifying groups include, but are not limited to,water-soluble and -insoluble polymers, therapeutic moieties, diagnosticmoieties, biomolecules. The locus of functionalization with themodifying group is selected such that it does not prevent the “modifiedsugar” from being added enzymatically to a peptide or glycosyl residueof the peptide.

Variant SAP Polypeptides

In part, the disclosure provides variant Serum Amyloid P (SAP)polypeptides. In particular, SAP variants of the invention includeglycosylated human SAP polypeptides that comprise one or more N-linkedor O-linked oligosaccharide chains each independently having one, two,three, four, five or more branches terminating with an α2,3-linkedsialic acid moiety. In some embodiments, all the branches of theN-linked or O-linked oligosaccharide chains terminate in α2,3-linkedmoieties. Other SAP variants of the invention include glycosylated humanSAP polypeptides that contain an N-linked or O-linked oligosaccharidechains having at least 20%, 25%, 30%, 35% 40%, 45%, 50%, 55%, 60%, 65%75%, 80%, 85%, or even at least 95% fewer α2,6-linked sialic acidmoieties than a wild-type SAP polypeptide derived from human serum. Insome embodiments, the N-linked or O-linked oligosaccharide chains aresubstantially free of α2,6-linked sialic acid moieties, e.g., havingless than 80%, 85%, 90%, 95%, 97%, 98% or even less than 99% α2,6-linkedsialic acid moieties relative to a wild-type SAP polypeptide derivedfrom human serum). Glycovariant SAP polypeptides of the invention maycomprise an N-linked oligosaccharide or O-linked chain having one ormore branches (e.g., having a bi-antennary, tri-antennary,tetra-antennary, penta-antennary, etc. structure). In certainembodiments, SAP polypeptides of the invention comprise an N-linked orO-linked oligosaccharide chain wherein one, two, three, four, or fivebranches of the oligosaccharide chain are substantially free ofgalactose and N-acetylglucosamine (e.g., having less than 80%, 85%, 90%,95%, 97%, 98% or even less than 99% N-acetylglucosamine relative to awild-type SAP polypeptide derived from human serum). Certain SAPpolypeptides of the invention have N-linked or O-linked oligosaccharidechains that are substantially free of galactose and N-acetylglucosamine(e.g., having less than 80%, 85%, 90%, 95%, 97%, 98% or even less than99% galactose and/or N-acetylglucosamine relative to a wild-type SAPpolypeptide derived from human serum). In some embodiments, SAPpolypeptides of the invention comprise an N-linked or O-linkedoligosaccharide chain wherein one, two, three, four, or five branches ofthe oligosaccharide chain contain one or more mannose residues. Incertain embodiments, the SAP polypeptide of the invention comprises anN-linked oligosaccharide having a pentasaccharide core ofMan[(α1,6-)-(Man(α1,3)]-Man(β1,4)-GlcNAc(β1,4)-GlcNAc(β1,N)-Asn. Thispentasaccharide core also may comprise one or more fucose or xyloseresidues. In certain embodiments, SAP polypeptides of the inventioncomprise an N-linked oligosaccharide chain wherein one, two, three,four, or five branches of the oligosaccharide chain have the structureNeuNAc2α3Galβ34GlcNAcβ32Mana6. SAP polypeptides of the invention alsomay comprise an N-linked oligosaccharide chain wherein all branches havethe structure NeuNAc2α3Galβ34G1cNAcβ32Manα6.

Variant SAP polypeptides of the invention may comprise one or more“modified” sugar residues. Modified sugars are substituted at anyposition that allows for the attachment of the modifying moiety orgroup. In preferred aspects, modified sugar is substituted at a positionthat still allows the sugar to function as a substrate for an enzymeused to couple the modified sugar to the SAP peptide. A modifying groupcan be attached to a sugar moiety by enzymatic means, chemical means ora combination thereof, thereby producing a modified sugar, e.g.,modified galactose, fucose, or sialic acid. Modifying groups suitablefor use in the present invention as well as methods for conjugatingthese modifying groups to sugar residues are described in the followingsection.

In preferred aspects, variant SAP polypeptides of the invention have anIC₅₀ for inhibiting the differentiation of monocytes into fibrocytes invitro that is less than one-half that of a corresponding sample ofwild-type SAP isolated from human serum. In some embodiments, variantSAP polypeptides of the invention have an IC₅₀ for inhibiting thedifferentiation of monocytes into fibrocytes in vitro that is less than⅓, less than ¼, less than 1/10, or less than 1/100 that of acorresponding sample of wild-type SAP isolated from human serum.

Variant SAP polypeptides of the invention may be at least 60%, at least70%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 1, as determined using the FASTDBcomputer program based on the algorithm of Brutlag et al. (Comp. App.Biosci., 6:237-245 (1990)). In a specific embodiment, parametersemployed to calculate percent identity and similarity of an amino acidalignment comprise: Matrix=PAM 150, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, GapPenalty=5 and Gap Size Penalty=0.05.

The term “SAP polypeptide” encompasses functional fragments and fusionproteins comprising any of the preceding. Generally, an SAP polypeptidewill be designed to be soluble in aqueous solutions at biologicallyrelevant temperatures, pH levels and osmolarity. The SAP protomers thatnon-covalently associate together to form a pentameric SAP complex mayhave identical amino acid sequences and/or post-translationalmodifications or, alternatively, individual SAP protomers within asingle complex may have different sequences and/or modifications. Theterm SAP polypeptide includes polypeptides comprising any naturallyoccurring SAP polypeptide as well as any variant thereof (includingmutants, fragments, and fusions). A SAP polypeptide of the invention maybe a recombinant polypeptide. In preferred embodiments, the SAPpolypeptide of the invention is a human SAP polypeptide.

In some embodiments, pharmaceutical compositions are provided comprisinga variant SAP polypeptide of the invention, or a functional fragmentthereof In some aspects, the amino acid sequence of a SAP variant maydiffer from SEQ ID NO: 1 by one or more conservative or non-conservativesubstitutions. As used herein, “conservative substitutions” are residuesthat are physically or functionally similar to the correspondingreference residues, i.e., a conservative substitution and its referenceresidue have similar size, shape, electric charge, and/or chemicalproperties (e.g., the ability to form covalent or hydrogen bonds).Preferred conservative substitutions are those fulfilling the criteriadefined for an accepted point mutation in Dayhoff et al., Atlas ofProtein Sequence and Structure 5:345-352 (1978 & Supp.). Examples ofconservative substitutions are substitutions within the followinggroups: (a) valine, glycine; (b) glycine, alanine; (c) valine,isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine,glutamine; (f) serine, threonine; (g) lysine, arginine, methionine; and(h) phenylalanine, tyrosine. Additional guidance concerning which aminoacid changes are likely to be phenotypically silent can be found inBowie et al., Science 247:1306-1310 (1990).

Variant SAP polypeptides and fragments thereof that retain biologicalfunction are useful in the pharmaceutical compositions and methodsdescribed herein. In some embodiments, a variant SAP polypeptide orfragment thereof binds FcγRI, FcγRIIA, and/or FcγRIIIB In someembodiments, a variant SAP polypeptide or fragment thereof inhibits oneor more of fibrocyte, fibrocyte precursor, myofibroblast precursor,and/or hematopoietic monocyte precursor differentiation. SAP variantsmay be generated by modifying the structure of an SAP polypeptide forsuch purposes as enhancing therapeutic efficacy or stability (e.g., exvivo shelf life and resistance to proteolytic degradation in vivo).

In certain aspects, the variant SAP polypeptides of the disclosure mayfurther comprise post-translational modifications in addition to anythat are naturally present in the SAP polypeptide. Such modificationsinclude, but are not limited to, acetylation, carboxylation,glycosylation (e.g., O-linked oligosaccharides, N-linkedoligosaccharides, etc.), phosphorylation, and lipidation. As a result,the modified SAP polypeptide may contain non-amino acid elements, suchas polyethylene glycols, lipids, poly- or mono-saccharides, andphosphates.

In certain aspects, one or more modifications to the SAP polypeptidedescribed herein may enhance the stability of the SAP polypeptide. Forexample, such modifications may enhance the in vivo half-life of the SAPpolypeptide or reduce proteolytic degradation of the SAP polypeptide.

In certain aspects, variant SAP polypeptides of the invention includefusion proteins having at least a portion of the human SAP polypeptideand one or more fusion domains or heterologous portions. Well knownexamples of such fusion domains include, but are not limited to,polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin,protein A, protein G, and immunoglobulin heavy chain constant region(Fc), maltose binding protein (MBP), or human serum albumin. A fusiondomain may be selected so as to confer a desired property. For example,some fusion domains are particularly useful for isolation of the fusionproteins by affinity chromatography. For the purpose of affinitypurification, relevant matrices for affinity chromatography, such asglutathione-, amylase-, and nickel-, or cobalt-conjugated resins areused. As another example, a fusion domain may be selected so as tofacilitate detection of the SAP polypeptides. Examples of such detectiondomains include the various fluorescent protein (e.g., GFP) as well as“epitope tags,” which are usually short peptide sequences for which aspecific antibody is available. Well known epitope tags for whichspecific monoclonal antibodies are readily available include FLAG,influenza virus hemagglutinin (HA) and c-myc tags. In some cases, thefusion domains have a protease cleavage site that allows the relevantprotease to partially digest the fusion proteins and thereby liberatethe recombinant protein therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation. In some cases, the SAP polypeptide may be fused to aheterologous domain that stabilizes the SAP polypeptide in vivo. By“stabilizing” is meant anything that increases serum half-life,regardless of whether this is because of decreased destruction,decreased clearance by the kidney, or other pharmacokinetic effect.Fusions with the Fc portion of an immunoglobulin and serum albumin areknown to confer increased stability.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an SAP polypeptide may be placed C-terminalto a heterologous domain, or, alternatively, a heterologous domain maybe placed C-terminal to an SAP polypeptide. The SAP polypeptide and theheterologous domain need not be adjacent in a fusion protein, andadditional domains or amino acid sequences (e.g., linker sequences) maybe included C- or N-terminal to either domain or between the domains.

Methods of Producing Altered N-Glycosylation Molecules

Described herein are methods of producing variant human SAPpolypeptides. The methods generally involve a step of contacting an SAPpolypeptide with one or more chemical or enzymatic agents to produce ormodify a glycosylation structure on the SAP polypeptide. The methods canbe cell-based or non-cell based.

Enzymes useful for producing or modifying glycan structures are wellknown in the art. Most enzymes/proteins useful in the methods of thedisclosure can be categorized into one of two functional classes:glycosyltransferases and glycosidases. Glycosyltransferases (e.g., N-acetylglucosaminyl-transferases, galactosyl-transferases,fucosyl-transferases, sialyl-transferases, glucosyl-transferases,mannosyl-transferases, etc.), as used herein, refers to anyenzyme/protein that has the ability to transfer a donor sugar to anacceptor moiety. Glycosidases (e.g., glucosidases, mannosidases,N-acetylglucosaminidases, sialidases, fucosidases, etc.), as usedherein, refers to any enzyme/protein that has the ability to catalyzethe hydrolysis of the glycosidic linkage between sugar moieties.

Cell-based methods for producing altered glyco-forms of an SAPpolypeptide use either wild-type (e.g., CHO cells) or geneticallyengineered cells that have at least one modified glycosylation activityrelative to a human cell. Cells suitable for the methods of thedisclosure include, for example, fungal cells, prokaryotic cell (i.e.,bacteria, Archaea) plant cells, or animal cells (e.g., nematode, insect,plant, bird, reptile, or mammal (e.g., a mouse, rat, rabbit, hamster,gerbil, dog, cat, goat, pig, cow, horse, whale, monkey, or human)). Thecells can be primary cells, immortalized cells, or transformed cells.Such cells can be obtained from a variety of commercial sources andresearch resource facilities, e.g., the American Type Culture Collection(Rockville, Md.). In certain aspects, the cell used for producing avariant SAP polypeptide is a CHO cell.

The term “glycosylation activity” refers to any activity that is (i)capable of adding N-linked or O-linked glycans to a target molecule(i.e., an oligosaccharyl-transferase activity); (ii) removing N-linkedor O-linked glycans from a target molecule; (iii) modifying one or moreN-linked or O-linked glycans on a target molecule; (iv) modifyingdolichol-linked oligosaccharides; (v) capable of aiding the activity ofone or more of the activities under i-iv. Accordingly, glycosylationactivity includes, for example, glycosidase activity,glycosyltransferase activity, sugar nucleotide synthesis, modification,or transporter activity. Modification of one or more N-linked orO-linked glycans on a target molecule includes the action of amannosylphosphoryl-transferase activity, a kinase activity, or aphosphatase activity, e.g., a mannosylphosphoryl-transferase, a kinase,or a phosphatase activity that alters the phosphorylation state ofglycans on target molecules.

Engineered cells useful in the methods of the disclosure may have one ormore genetic modifications including, but not limited to: (i) deletionof an endogenous gene encoding a protein having glycosylation activity;(ii) introduction of a recombinant nucleic acid encoding a mutant formof a protein (e.g., endogenous or exogenous protein) having anglycosylation activity; (iii) introduction or expression of an RNAmolecule that interferes with the functional expression of a proteinhaving the glycosylation activity; (iv) introduction of a recombinantnucleic acid encoding a wild-type (e.g., endogenous or exogenous)protein having glycosylation activity; or (v) altering the promoter orenhancer elements of one or more endogenous genes encoding proteinshaving glycosylation activity to thus alter the expression of theencoded proteins. RNA molecules described above include, for example,small-interfering RNA (siRNA), short hairpin RNA (shRNA), anti-senseRNA, or micro RNA (miRNA). It is understood that item (ii) includes, forexample, replacement of an endogenous gene (e.g., by homologousrecombination) with a gene encoding a protein having greaterglycosylation activity relative to the endogenous gene so replaced.

The genetically engineered cells described herein have one or morealtered glycosylation activities such as: (i) an increase in one or moreglycosylation activities in the genetically modified cell, (ii) adecrease in one or more glycosylation activities in the geneticallymodified cell, (iii) a change in the localization or intracellulardistribution of one or more glycosylation activities in the geneticallymodified cell, or (iv) a change in the ratio of one or moreglycosylation activities in the genetically modified cell relative to anunmodified cell of the same origin. It is understood that an increase inthe amount of glycosylation activity can be due to overexpression of oneor more proteins having glycosylation activity, an increase in copynumber of an endogenous gene (e.g., gene duplication), or an alterationin the promoter, enhancer, or suppressor of an endogenous gene thatstimulates an increase in expression of the protein encoded by the gene.A decrease in one or more glycosylation activities can be due tooverexpression of a mutant form (e.g., a dominant negative form) of oneor more proteins having glycosylation -altering activities, introductionor expression of one or more interfering RNA molecules that reduce theexpression of one or more proteins having an glycosylation activity, ordeletion of one or more endogenous genes that encode a protein havingglycosylation activity.

Genetically engineered cells used by the methods of the disclosure canexpress (e.g., overexpress) wild-type or mutant genes encoding proteinshaving glycosylation activity. Such genes include, but are not limitedto, ALG7, ALG13, ALG14, ALG1, ALG2, ALG11, RFT1, ALG3, ALG9, ALG12,ALG6, ALG8, ANL1, ALG10, ALG5, OST3, OST4, OST6, STT3, OST1, OST5, WBP1,SWP1, OST2, DPM1, SEC59, OCH1, MNN9, VAN1, MNN8, MNN10, MNN11, HOC1,MNN2, MNN5, MNN6, KTR1, YUR1, MNN4, KRE2, KTR2, KTR3, MNN1, MNS1, MNN4,PNO1, MNN9, glucosidase I, glucosidase II, or endomannosidase. Genesencoding proteins having glycosylation activity can be from any species(e.g., lower eukaryotes (e.g., fungus (including yeasts) ortrypanosomes), prokaryotes (i.e., bacteria or Archaea), plant, or animal(e.g., insect, bird, reptile, or mammal, such as mouse or rat, dog, cat,horse, goat, cow, pig, non-human primate, or human). It is understoodthat genetically engineered cells used for the methods of the disclosurecan express any number of genes (e.g., genes encoding proteins havingglycosylation activity) and/or any combination of one or more (e.g., 2,3, 4, 5,6, 7, 8, 9 10, 11, 12, 15, or 20 or more) of any of the genesrecited herein. In addition, any genetically engineered cells used bythe methods of the disclosure may comprise any number of mutations thatalter or abrogate one or more glycosylation activities.

In some embodiments, the term “gene expression” or “expression” refersto the cellular processes by which a biologically active polypeptide isproduced from a DNA sequence and exhibits a biological activity in acell. As such, gene expression involves the processes of transcriptionand translation, but also involves post-transcriptional andpost-translational processes that can influence a biological activity ofa gene or gene product. These processes include, but are not limited to,RNA syntheses, processing, and transport, as well as polypeptidesynthesis, transport, and post-translational modification ofpolypeptides.

For example, the disclosure provides methods for making an SAPpolypeptide of the invention by expressing a SAP gene in a cell. In someembodiments, the cell may contain an endogenous SAP gene or fragmentthereof. In other embodiments, a polynucleotide coding for an exogenousSAP polypeptide or fragment thereof may be introduced (e.g.,transformed, transfected, etc.) into a cell. Suitable SAPpolynucleotides that can be introduced into a cell include nucleic acidfragments as well as nucleic acid constructs or expression vectors. Inpreferred embodiments, the endogenous or exogenous gene encodes a humanSAP polypeptide.

In some embodiments, the nucleic acid fragment, e.g., encoding a humanSAP polypeptide, used to transform the host cell may include aShine-Dalgarno site (e.g., a ribosome binding site) and a start site(e.g., the codon ATG) to initiate translation of the transcribed messageto produce the enzyme. It may, also include a termination sequence toend translation. A termination sequence is typically a codon for whichthere exists no corresponding aminoacetyl-tRNA, thus ending polypeptidesynthesis. In some embodiments, a nucleic acid construct encoding an SAPpolypeptide can be delivered, for example, as an expression plasmidthat, when transcribed in the cell, produces as SAP polypeptide.

In some embodiments, a suitable expression vector comprises a nucleotidesequence encoding an SAP polypeptide of the invention operably linked toat least one regulatory sequence. Regulatory sequences areart-recognized and are selected to direct expression of any of thepolypeptides of the disclosure. Accordingly, the term regulatorysequence includes promoters, enhancers and other expression controlelements. Exemplary regulatory sequences are described in Goeddel; GeneExpression Technology: Methods in Enzymology, Academic Press, San Diego,Calif. (1990). For instance, any expression control sequence thatregulates the expression of a DNA sequence when operatively linked to itmay be used in these vectors to express any of the polypeptides of thedisclosure. Such useful expression control sequences, include, forexample, the early and late promoters of SV40, tet promoter, adenovirusor cytomegalovirus immediate early promoter, the lac system, the trpsystem, the TAC or TRC system, T7 promoter whose expression is directedby T7 RNA polymerase, the major operator and promoter regions of phagelambda , the control regions for fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase, e.g., Pho5, the promoters of the yeast a-matingfactors, the polyhedron promoter of the baculovirus system and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof. Itshould be understood that the design of the expression vector may dependon such factors as the choice of the host cell to be transformed and/orthe type of protein desired to be expressed. Moreover, the vector's copynumber, the ability to control that copy number and the expression ofany other protein encoded by the vector, such as antibiotic markers,should also be considered.

In some embodiments, the nucleic acid fragment or expression system usedto transform the host cell optionally may include one or more markersequences. Generally speaking, suitable marker sequences typicallyencode a gene product, usually an enzyme that inactivates or otherwisedetects or is detected by a compound in the growth medium. For example,the inclusion of a marker sequence may render the transformed cellresistant to an antibiotic, or it may confer compound-specificmetabolism on the transformed cell. Examples of suitable markersequences that confer resistance include kanamycin, ampicillin,chloramphenicol and tetracycline. Alternatively, rather than selectivepressure, a marker gene may be used that allows for detection ofparticular colonies containing the gene, such as β-galactosidase, wherea substrate is employed that provides for a colored product.

A variety of methods are suitable for transforming a cell of the presentinvention with a nucleic acid fragment or expression vector. Commontransformation methods include electroporation, liposomal-mediatedtransformation, calcium-mediated transformation, and viral-mediatedtransfection.

In certain aspects, when a host cell is transformed with a nucleic acidfragment or expression system of the present invention, the gene (e.g.,human SAP) in said system can be integrated into the chromosomal DNA ofthe host cells by a so-called homologous recombination and theexpression system will be carried stably in the host.

In order to integrate the expression system in the vector intochromosomal DNA of the host cells, an appropriate selection marker genemay be used wherein said marker gene has a sequence homologous to thegene on chromosomal DNA of the specific host cell. Selection markers forsuch a purpose can be easily selected by a skilled person. As anexample, a preferred marker is a gene that exists on a chromosome andrelates to the metabolism of the host cells. Namely, it is preferred touse a host which has been modified in such a manner that theabove-mentioned gene on the chromosome will be inactivated by anappropriate means such as a mutation. The host can then be subjected toa homologous recombination with an expression vector containing thecorresponding intact gene, whereupon only transformants which containthe normal metabolism gene can grow to be selected. Therefore, if such amarker gene has been introduced to the expression vector, a homologousrecombination will take place between the marker gene in said expressionvector and the corresponding portion of the chromosomal DNA, whereby theexpression cassette of the heterologous gene will simultaneously beintegrated into the chromosomal DNA.

In some embodiments, the term “expressing” a protein in a cell alsoincludes methods of introducing a protein itself into cells. Therefore,in certain aspects, the disclosure provides methods for making an SAPpolypeptide of the invention by introducing an SAP polypeptide into acell. Techniques for introducing polypeptides directly into a cell areknown in the art and generally involve a process of cell membranepermeation. Such techniques include, but are not limited to,microinjection of SAP polypeptides; encapsulating an SAP polypeptidewithin membrane vesicles (e.g., liposomes, capsular bodies, erythrocyteghosts, protoplasts, etc.) and contacting them with a cell membrane tothereby cause intracellular introduction of the SAP polypeptide byfusion; using physical methods (e.g., mechanical, chemical, electrical,etc.) that rely on macromolcules entering cells by diffusion throughholes transiently introduced in their plasma membranes (e.g.,scrape-loading, bead-loading, etc.); and by uptake through naturalendocytosis owing to cellular phagocytosis. A method of intracellularintroduction may utilize a receptor-mediated pathway, wherein one ofvarious receptors expressed on the cell surface is set as a target andan SAP polypeptide is attached (covalently or non-covalently) to thecognate ligand that acts as a carrier moiety. Several methods have beendescribed for introducing proteins into living cells using electrostaticadsorption, wherein the protein is first cationized and then contactedwith the negatively charged surface of a cell (See Japanese PatentPublication No. 2004/049214).

In certain aspects, the disclosure provides CHO cells that express a SAPpolypeptide. In some embodiments, the CHO cell expresses an exogenousSAP polypeptide. In preferred embodiments, the CHO cell expresses ahuman SAP polypeptide. In some embodiments, the disclosure provides CHOcells comprising a polynucleotide sequence encoding an SAP polypeptide.In preferred embodiments, the polynucleotide sequence encodes a humanSAP polypeptide. Any of the aforementioned techniques may be used to“express” an SAP polypeptide of the invention in a CHO cell or any othersuitable cell disclosed herein.

Where any of the glycosylation activities of the wild-type orgenetically engineered cell are inducible or conditional on the presenceof an inducing cue (e.g., a chemical or physical cue), the wild-type orgenetically engineered cell can, optionally, be cultured in the presenceof an inducing agent before, during, or subsequent to the introductionof the nucleic acid encoding a SAP polypeptide or a SAP polypeptide. Forexample, following introduction of the SAP polypeptide into the cell canbe exposed to a chemical inducing agent that is capable of promoting theexpression of one or more proteins having a wild-type or alteredN-glycosylation activity. Where multiple inducing cues induceconditional expression of one or more proteins having wild-type and/oraltered N-glycosylation activity, a cell can be contacted with multipleinducing agents. In some embodiments, the culture medium may be modifiedto produce the desired glycan structure on the SAP polypeptide. Forexample, the serum, glucose, and/or lipid (e.g., dolichol) concentrationof the medium may be modified for optimal production of the desired SAPglycovariant. In some embodiments, the temperature, pH, and/orosmolarity of culture medium may be altered for optimal production ofthe desired SAP glycovariant.

A variant SAP polypeptide of the invention can be further processed invivo or can be processed in vitro prior to or following isolation fromthe cell or cell medium. The further processing can include modificationof one or more glycan residues of the SAP polypeptide or modification tothe SAP polypeptide other than to its glycan structure(s). In someembodiments, the additional processing of the SAP polypeptide caninclude the addition (covalent or non-covalent joining) of one or moreheterologous moieties. In some embodiments, the further processinginvolves enzymatic or chemical treatment of the SAP polypeptide.Enzymatic treatment can involve contacting the SAP polypeptide with oneor more glycosidase, phosphodiesterase, phospholipase,glycosyltransferase, or protease for a time sufficient to induce themodification, addition, or deletion of glycan residues (e.g., galactose,mannose, fucose, sialic acid, xylose, N-acetylglucosamine, etc.) on theSAP polypeptide. Customization of an N-linked oligosaccharide chain maybe accomplished by the sequential modification, addition, or deletion ofthe desired sugar moieties, using techniques well known in the art.Enzymatic cleavage of carbohydrate moieties on peptide variants can beachieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., 1987, Meth. Enzymol. 138:350. Exemplarymethods of adding sugar moieties are disclosed in U.S. Pat. Nos.5,876,980, 6,030,815, 5,728,554, and 5,922,577.

In certain embodiments, modification of the SAP glycan structurerequires the presence of one or more sugar nucleotides. Exemplary sugarnucleotides that are used in the present invention include nucleotidemono-, di- or triphosphates or analogs thereof In a preferredembodiment, the modified sugar nucleotide is selected from aUDP-glycoside, CMP-glycoside, or a GDP-glycoside. Even more preferably,the sugar nucleotide is selected from an UDP-galactose,UDP-galactosamine, UDP-glucose, UDP-glucosamine, GDP-mannose,GDP-fucose, CMP-sialic acid, CMP-N-glycolylneuraminic acid or CMP-NeuAc.In certain embodiments, a modified sugar nucleotide is utilized to add amodified sugar residue to the SAP polypeptide. N-acetylamine derivativesof the sugar nucleotides are also of use in the method of the invention.

Chemical addition or removal of glycosyl moieties can be carried out byany suitable method. For example, chemical deglycosylation may involveexposure of the SAP polypeptide to trifluoromethanesulfonic acid, oranother strong acid. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the peptide intact. Methods ofchemical deglycosylation are described by Haldmuddin et al., 1987, Arch.Biochem. Biophys. 259: 52 and by Edge et al., 1981, Anal. Biochem. 118:131. Chemical treatment can, for example, involve contacting the alteredSAP polypeptide with an acid, such as hydrofluoric acid, for a timesufficient to induce modification of the SAP polypeptide. Hydrofluoricacid treatment, under certain conditions, specifically removes themannose residues that are phosphodiester-linked to glycans, whileleaving the phosphate on the glycan. An altered SAP polypeptide can befurther processed by addition or removal of a phosphate group from oneor more N-glycans. For example, an altered SAP polypeptide can becontacted with a mannosyl kinase or a mannosyl phosphatase.

In certain aspects, it is desirable to modify only terminal sugarmoieties on the SAP polypeptide glycan structure (N-linked or O-linkedoligosaccharide). In some embodiments, one or more branches of the SAPglycan structure are modified by the addition, removal, substitution ormodification of at least one terminal sialic acid residue. Suitablemethods for modifying glycans described herein may be used to alter onlythe terminal sugar moiety on the SAP glycan structure. In someembodiments, a terminal sialic acid residue having an α2,6-linkage,α2,8-linkage, or α2,9-linkage is replaced with one or more terminalα2,3-linked sialic acid residue. In a particular aspect, human SAPcomprising terminal α2,6-linked sialic residues is modified according toone of methods of the disclosure in order to replace one or more of theterminal α2,6-linked sialic residues with one or more α2,3-linked sialicacid residues. In some embodiments, a terminal α2,3-linked sialic acidresidue is modified to add one or more α2,6-linked, α2,8-linked, and/orα2,9-linked sialic acid residues.

In certain aspects, the process of making an SAP polypeptide of theinvention involves a first step of deglycosylating the SAP polypeptide.The SAP polypeptide may be partially or fully deglycosylated. In someembodiments, the first step of deglycosylation involves removing onlyterminal sugar moieties from at least one branch of the glycan structureon the SAP polypeptide. In some embodiments, the first step ofdeglycosylation removes at least one α2,6-linked sialic acid residue. Insome embodiments, the first step of deglycosylation removes at least oneO-linked glycan. In some embodiments, the first step of deglycosylationremoves at least one N-linked glycan. In some embodiments, the firststep of deglycosylation removes all O-linked and N-linked glycans. Insome embodiments, a deglycosylated SAP polypeptide (partially or fully)is further processed according to the methods of the disclosure, whichincludes but is not limited to, enzymatically or chemically modifyingthe partially or fully deglycosylated SAP polypeptide, introducing thepartially or fully deglycosylated SAP polypeptide into a cell, orcombination thereof, wherein the method(s) produce a variant SAPpolypeptide of the invention. In some embodiments, after a partially orfully deglycosylated SAP polypeptide has been introduced into a cell, itmay be further modified, in vivo or in vitro, according to the methodsof the disclosure to produce a variant SAP polypeptide of the invention.Similarly, a partial or fully deglycosylated SAP polypeptide that ismodified in vitro, using enzymatic or chemical methods described herein,may be introduced into a cell to produce a variant SAP polypeptide ofthe invention.

It is understood that an SAP polypeptide of the invention can be, butneed not be, processed in a cell. For example, the disclosure alsoprovides cell-free methods of producing a variant SAP polypeptide of theinvention. In some aspects the cell-free methods include the step ofcontacting an SAP polypeptide, under glycosylation conditions, with acell lysate prepared from a wild-type cell (e.g., a fungal cell, a plantcell, or an animal cell) or genetically engineered cell having at leastone modified glycosylation activity, wherein contacting the SAPpolypeptide with the cell lysate attaches an N-linked or O-linkedoligosaccharide to the SAP polypeptide or modifies an existing N-linkedor O-linked oligosaccharide on the SAP polypeptide. By “N-glycosylationconditions” is meant that a mixture (e.g., of SAP polypeptide and celllysate) is incubated under conditions that allow for the desired alteredN-glycosylation.

Suitable methods for obtaining cell lysates that preserve the activityor integrity of one or more glycosylation activities in the lysate caninclude the use of appropriate buffers and/or inhibitors, includingnuclease, protease and phosphatase inhibitors that preserve or minimizechanges in N-glycosylation activities in the cell lysate. Suchinhibitors include, for example, chelators such asethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(P-aminoethylether) N,N,N1,N1-tetraacetic acid (EGTA), protease inhibitors such asphenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, antipain,and phosphatase inhibitors such as phosphate, sodium fluoride, andvanadate. Inhibitors can be chosen such that they do not interfere withor only minimally adversely affect the N-glycosylation activity, oractivities, of interest. Appropriate buffers and conditions forobtaining lysates containing enzymatic activities are described in,e.g., Ausubel et al. Current Protocols in Molecular Biology (Supplement47), John Wiley & Sons, New York (1999); Harlow and Lane, Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press (1988); Harlow andLane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press(1999); Tietz Textbook of Clinical Chemistry, 3rd ed. Burtis andAshwood, eds. W. B. Saunders, Philadelphia, (1999).

A cell lysate can be further processed to eliminate or minimize thepresence of interfering substances, as appropriate. If desired, a celllysate can be fractionated by any of a variety of methods well known tothose skilled in the art, including subcellular fractionation, andchromatographic techniques such as ion exchange, hydrophobic and reversephase, size exclusion, affinity, and hydrophobic charge-inductionchromatography (see, e.g., Scopes, Protein Purification: Principles andPractice, third edition, Springer-Verlag, New York (1993); Burton andHarding, J. Chromatogr. A 814:71-81 (1998)), or any other suitablepurification technique.

A cell lysate can be prepared in which whole cellular organelles remainintact and/or functional. For example, a lysate can contain one or moreof intact rough endoplasmic reticulum, intact smooth endoplasmicreticulum, or intact Golgi apparatus. Suitable methods for preparinglysates containing intact cellular organelles and testing for thefunctionality of the organelles are described in, e.g., Moreau et al.(1991) J. Biol. Chem. 266(7):4329-4333; Moreau et al. (1991) J. Biol.Chem. 266(7):4322-4328; Rexach et al. (1991) J. Cell Biol.114(2):219-229; and Paulik et al. (1999) Arch. Biochem. Biophys.367(2):265-273; the disclosures of each of which are incorporated hereinby reference in their entirety.

The SAP polypeptide can be contacted with just one purified proteinhaving glycosylation activity. In some embodiments, the SAP polypeptidecan be contacted with more than one purified proteins havingglycosylation activity. One or more proteins having glycosylationactivity can be purified using standard protein isolation techniques. AnSAP polypeptide can be contacted with one or more proteins in a suitablebuffer for a time sufficient to induce modification of the SAPpolypeptides as described above. The SAP polypeptide can be contactedwith more than one protein at the same time or sequentially. Where theSAP polypeptide is contacted sequentially to more than one proteinhaving glycosylation activity, the SAP polypeptide can, but need not, bepurified after one or more steps. That is, an SAP polypeptide can becontacted with protein activity “A” and then purified before contactingthe molecule to protein activity “B”. Methods of modifying peptides assuch are known in the art, e.g., Lee and Park (2002) 30(6):716-720 andFujita and Takegawa (2001) Biochem. Biophys. Res. Commun.282(3):678-682, the disclosures of which are incorporated herein byreference in their entirety.

In certain aspects, the methods of the disclosure comprise a step ofisolating an SAP polypeptide, e.g., from a cell or from components of acell lysate. Many standard techniques for protein isolation are known inthe art. For example, methods of isolating polypeptides include, but arenot limited to, size-exclusion chromatography, reverse-phasechromatography, liquid chromatography (e.g., HPLC), affinitychromatography (e.g., metal chelation or immunoaffinity chromatography),ion-exchange chromatography, hydrophobic-interaction chromatography,precipitation, differential solubilization, immunoprecipitation,centrifugation (e.g., ultracentrifugation, sucrose gradientcentrifugation, etc.) or any combination thereof In some embodiments,the SAP polypeptide may be conjugated to an affinity tag to facilitatepurification of the polypeptide. Suitable affinity tags for SAPpurification include, but are not limited to, chitin binding protein(CBP), maltose binding protein (MBP), glutathione-S-transferase (GST),streptavidin, biotin, and poly(His) tags. Affinity tags may be producedas part of the recombinant protein (i.e., as a fusion protein comprisinga heterologous affinity tag domain and an SAP polypeptide domain) orattached (covalently or non-covalently) in vivo or in vitro to the SAPpolypeptide. In some embodiments, multiple steps of purification can beused to isolate an SAP polypeptide. For example, an SAP polypeptidecomprising a purification tag can be affinity-purified from a celllysate or multi-component mixture using affinity purification. Then theaffinity-purified SAP polypeptide can be further purified to remove anyminor unwanted contaminants by an additional purification step, e.g.,size exclusion chromatography or RP-HPLC. Where a polypeptide of theinvention is produced in a cell, the SAP polypeptide can be isolatedfrom the cell itself or from the media in which cell was cultured. Insome embodiments, SAP polypeptides of the invention are produced andsecreted from a cell into the culture media. In these embodiments,isolation may comprise separation of the cellular fraction from thesoluble, SAP-containing fraction (e.g., by centrifugation).

In some aspects, it can be advantageous to link the SAP polypeptide to asolid-phase support prior to contacting the target molecule with one ormore N-glycosylation activities. Such linkage can allow for easierpurification following the N-glycosylation modifications. Suitablesolid-phase supports include, but are not limited to, multi-well assayplates, particles (e.g., magnetic or encoded particles), achromatography column, or a membrane.

In some embodiments, any of the altered SAP polypeptides describedherein can be attached to a heterologous moiety using enzymatic orchemical means. A “heterologous moiety” refers to any constituent thatis joined (e.g., covalently or non-covalently) to the altered targetmolecule, which constituent is different from a constituent originallypresent on the SAP polypeptide. Heterologous moieties include, forexample, water-soluble and—insoluble polymers, targeting moieties,therapeutic moieties, diagnostic moieties, and biomolecules.

SAP polypeptides of the invention may comprise one or more “modified”sugar residues. A modifying group can be attached to a sugar moiety byenzymatic means, chemical means or a combination thereof, therebyproducing a modified sugar, e.g., modified galactose, fucose, or sialicacid. When a modified sialic acid is used, either a sialyl-transferaseor a trans-sialidase can be used in these methods. The sugars may besubstituted at any position that allows for the attachment of themodifying moiety, yet which still allows the sugar to function as asubstrate for the enzyme used to couple the modified sugar to thepeptide.

In general, the sugar moiety and the modifying group are linked togetherthrough the use of reactive groups, which are typically transformed bythe linking process into a new organic functional group or unreactivespecies. The sugar reactive functional group(s) may be located at anyposition on the sugar moiety. Reactive groups and classes of reactionsuseful in practicing the present invention are generally those that arewell known in the art of bioconjugate chemistry. Currently favoredclasses of reactions available with reactive sugar moieties are thosewhich proceed under relatively mild conditions. These include, but arenot limited to nucleophilic substitutions (e.g., reactions of amines andalcohols with acyl halides, active esters), electrophilic substitutions(e.g., enamine reactions) and additions to carbon-carbon andcarbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alderaddition). These and other useful reactions are discussed in, forexample, Smith and March, Advanced Organic Chemistry, 5th Ed., JohnWiley & Sons, New York, 2001; Hermanson, Bioconjugate Techniques,Academic Press, San Diego, 1996; and Feeney et al., Modification ofProteins; Advances in Chemistry Series, Vol. 198, American ChemicalSociety, Washington, D.C., 1982.

Useful reactive functional groups pendent from a sugar nucleus ormodifying group include, but are not limited to: (a) carboxyl groups andvarious derivatives thereof (e.g., N-hydroxysuccinimide esters,N-hydroxybenzotriazole esters, acid halides, acyl imidazoles,thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromaticesters); (b) hydroxyl groups, which can be converted to, e.g., esters,ethers, aldehydes, etc.; (c) haloalkyl groups, wherein the halide can belater displaced with a nucleophilic group such as, for example, anamine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion,thereby resulting in the covalent attachment of a new group at thefunctional group of the halogen atom; (d) dienophile groups, which arecapable of participating in Diels-Alder reactions such as, for example,maleimido groups (e) aldehyde or ketone groups, such that subsequentderivatization is possible via formation of carbonyl derivatives suchas, for example, imines, hydrazones, semicarbazones or oximes, or viasuch mechanisms as Grignard addition or alkyllithium addition; (f)sulfonyl halide groups for subsequent reaction with amines, for example,to form sulfonamides; (e) thiol groups, which can be, for example,converted to disulfides or reacted with alkyl and acyl halides; (h)amine or sulfhydryl groups, which can be, for example, acylated,alkylated or oxidized; (i) alkenes, which can undergo, for example,cycloadditions, acylation, Michael addition, metathesis, Heck reaction,etc.; (j) epoxides, which can react with, for example, amines andhydroxyl compounds.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive sugar nucleus or modifying group. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., ProtectiveGroups in Organic Synthesis, John Wiley & Sons, New York, 1991.

In some embodiments, the modified sugar is an activated sugar. Activatedmodified sugars useful in the present invention are typically glycosideswhich have been synthetically altered to include an activated leavinggroup. As used herein, the term “activated leaving group” refers tothose moieties which are easily displaced in enzyme-regulatednucleophilic substitution reactions. Many activated sugars are known inthe art. See, for example, Vocadlo et al., In Carbohydrate Chemistry andBiology, Vol. 2, Ernst et al. Ed., Wiley-VCH Verlag: Weinheim, Germany,2000; Kodama et al., Tetrahedron Lett. 34: 6419 (1993); Lougheed, etal., J. Biol. Chem. 274: 37717 (1999)). Examples of such leaving groupsinclude fluoro, chloro, bromo, tosylate, mesylate, triflate and thelike. Preferred activated leaving groups for use in the presentinvention are those that do not significantly sterically encumber theenzymatic transfer of the glycoside to the acceptor. Accordingly,preferred embodiments of activated glycoside derivatives includeglycosyl fluorides and glycosyl mesylates, with glycosyl fluorides beingparticularly preferred. Among the glycosyl fluorides, α-galactosylfluoride, α-mannosyl fluoride, α-glucosyl fluoride, α-fucosyl fluoride,α-xylosyl fluoride, α-sialyl fluoride, α-N-acetylglucosaminyl fluoride,α-N-acetylglucosaminyl fluoride, β-galactosyl fluoride, β-mannosylfluoride, .beta.-glucosyl fluoride, β-fucosyl fluoride, β-xylosylfluoride, β-sialyl fluoride, β-N-acetylglucosaminyl fluoride andβ-N-acetylgalactosaminyl fluoride are most preferred.

In certain aspects, a modified sugar residue is conjugated to one ormore water-soluble polymers. Many water-soluble polymers are known tothose of skill in the art and are useful in practicing the presentinvention. The term water-soluble polymer encompasses species such assaccharides (e.g., dextran, amylose, hyaluronic acid, poly(sialic acid),heparans, heparins, etc.); poly(amino acids); nucleic acids; syntheticpolymers (e.g., poly(acrylic acid), poly(ethers), e.g., poly(ethyleneglycol)); peptides, proteins, and the like. The present invention may bepracticed with any water-soluble polymer with the sole limitation thatthe polymer must include a point at which the remainder of the conjugatecan be attached.

Methods and chemistry for activation of water-soluble polymers andsaccharides as well as methods for conjugating saccharides and polymersto various species are described in the literature. Commonly usedmethods for activation of polymers include activation of functionalgroups with cyanogen bromide, periodate, glutaraldehyde, bi-epoxides,epichlorohydrin, divinyl sulfone, carbodiimide, sulfonyl halides,trichlorotriazine, etc. (see, R. F. Taylor, (1991), ProteinImmobilization, Fundamentals and Applications, Marcel Dekker, N.Y.; S.S. Wong, (1992), Chemistry of Protein Conjugation and Crosslinking, CRCPress, Boca Raton; G. T. Hermanson et al., (1993), Immobilized AffinityLigand Techniques, Academic Press, N.Y.; Dunn, R. L., et al., Eds.Polymeric Drugs and Drug Delivery Systems, ACS Symposium Series Vol.469, American Chemical Society, Washington, D.C. 1991).

In certain aspects, a modified sugar residue is conjugated to one ormore water-insoluble polymers. In some embodiments, conjugation to awater-insoluble polymer can be used to deliver a therapeutic peptide ina controlled manner. Polymeric drug delivery systems are known in theart. See, for example, Dunn et al., Eds. Polymeric drugs and DrugDelivery Systems, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991. Those of skill in the art willappreciate that substantially any known drug delivery system isapplicable to the conjugates of the present invention.

Representative water-insoluble polymers include, but are not limited to,polyphosphazenes, poly(vinyl alcohols), polyamides, polycarbonates,polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, poly(methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate)polyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly (ethylene terephthalate), poly(vinyl acetate), polyvinylchloride, polystyrene, polyvinyl pyrrolidone, pluronics, andpolyvinylphenol, and copolymers thereof.

These and the other polymers discussed herein can be readily obtainedfrom commercial sources such as Sigma Chemical Co. (St. Louis, Mo.),Polysciences (Warrenton, Pa.), Aldrich (Milwaukee, Wis.), Fluka(Ronkonkoma, N.Y.), and BioRad (Richmond, Calif.), or else synthesizedfrom monomers obtained from these suppliers using standard techniques.Representative biodegradable polymers useful in the conjugates of theinvention include, but are not limited to, polylactides, polyglycolidesand copolymers thereof poly(ethylene terephthalate), poly(butyric acid),poly(valeric acid), poly(lactide-co-caprolactone),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, blends andcopolymers thereof. Of particular use are compositions that form gels,such as those including collagen, and pluronics.

In a preferred embodiment, one or more modified sugar residues areconjugated to one or more PEG molecules.

In certain aspects, the modified sugar is conjugated to a biomolecule.Biomolecule of the invention may include, but are not limited to,functional proteins, enzymes, antigens, antibodies, peptides, nucleicacids (e.g., single nucleotides or nucleosides, oligonucleotides,polynucleotides and single- and higher-stranded nucleic acids), lectins,receptors or a combination thereof. Some preferred biomolecules areessentially non-fluorescent, or emit such a minimal amount offluorescence that they are inappropriate for use as a fluorescent markerin an assay. Other biomolecules may be fluorescent.

In some embodiments, the biomolecule is a targeting moiety. A “targetingmoiety” and “targeting agent”, as used herein, refer to species thatwill selectively localize in a particular tissue or region of the body.In some embodiments, a biomolecule is selected to direct the SAPpolypeptide of the invention to a specific intracellular compartment,thereby enhancing the delivery of the peptide to that intracellularcompartment relative to the amount of underivatized peptide that isdelivered to the tissue. The localization is mediated by specificrecognition of molecular determinants, molecular size of the targetingagent or conjugate, ionic interactions, hydrophobic interactions and thelike. Other mechanisms of targeting an agent to a particular tissue orregion are known to those of still in the art.

In some embodiments, the modified sugar includes a therapeutic moiety.Those of skill in the art will appreciate that there is overlap betweenthe category of therapeutic moieties and biomolecules, i.e., manybiomolecules have therapeutic properties or potential.

Classes of useful therapeutic moieties include, for example,non-steroidal anti-inflammatory drugs; steroidal anti-inflammatorydrugs; adjuvants; antihistaminic drugs; antitussive drugs; antipruriticdrugs; anticholinergic drugs; anti-emetic and antinauseant drugs;anorexic drugs; central stimulant drugs; antiarrhythmic drugs;β-adrenergic blocker drugs; cardiotonic drugs; antihypertensive drugs;diuretic drugs; vasodilator drugs; vasoconstrictor drugs; antiulcerdrugs; anesthetic drugs; antidepressant drugs; tranquilizer and sedativedrugs; antipsychotic drugs; and antimicrobial drugs.

Other drug moieties useful in practicing the present invention includeantineoplastic drugs, cytocidal agents, anti-estrogens, andantimetabolites. Also included within this class are radioisotope-basedagents for both diagnosis (e.g., imaging) and therapy, and conjugatedtoxins.

The therapeutic moiety can also be a hormone, a muscle relaxant, anantispasmodic, bone activating agent, endocrine modulating agent,modulator of diabetes, androgen, antidiuretics, or calcitonin drug.

Other useful modifying groups include immunomodulating drugs,immunosuppressants, etc. Groups with anti-inflammatory activity, such assulindac, etodolac, ketoprofen and ketorolac, are also of use. Otherdrugs of use in conjunction with the present invention will be apparentto those of skill in the art.

The altered N-glycosylation SAP polypeptides produced by the methods ofthe disclosure can be homogeneous (i.e., the sample of SAP polypeptideis uniform in specific N-glycan structure) or substantially homogeneous.By “substantially homogeneous” is meant that at least about 25% (e.g.,at least about 27%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,or at least about 95%, or at least about 99%) of the SAP polypeptidescontain the same specific N-glycan structure.

In some embodiments, variant SAP polypeptides of the invention have anIC50 for inhibiting the differentiation of monocytes into fibrocytes invitro that is less than ½, less than ⅓, less than ¼, less than 1/10, orless than 1/100 that of a corresponding sample of wild-type SAP isolatedfrom human serum. There are many well characterized methods fordetermining the responsiveness of Peripheral Blood Mononuclear Cells(PBMCs) or monocyte cells to SAP for fibrocyte differentiation. Thesemethods may be used to determine the relative potency of any of the SAPvariant polypeptides of the invention in comparison to a sample of humanserum-derived SAP, any other SAP variant polypeptide, or other fibrocytesuppressant or activating agent. PBMCs or monocytes suitable for use inthese methods may be obtained from various tissue culture lines.Alternatively, suitable cells for fibrocyte differentiation assays maybe obtained from any biological sample that contains PBMC or monocytecells. The biological sample may be obtained from serum, plasma, healthytissue, or fibrotic tissue. In general, fibrocyte differentiation assaysare conducted by incubating PBMC or monocyte cells in media with variousconcentrations of a SAP polypeptide to determine the degree of fibrocytedifferentiation. The concentration of SAP can range from 0.0001 μg/mL to1 mg/ml, and in some embodiments is 0.001 μg/mL, 1.0 μg/mL, 5 μg/mL, 10μg/mL, 15 μg/mL, 20 μg/mL, 25 μg/mL, 30 μg/mL, 35 μg/mL, 40 μg/mL, 45μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, 300 μg/mL, or 500 μg/mL. In someassays, the media can be supplemented with between 1-100 ng/ml hMCSF;the preferred concentration of hMCSF being 25 ng/mL. The indication thatPBMC and monocytes have differentiated into fibrocytes can be determinedby one skilled in the art. In general, fibrocytes are morphologicallydefined as adherent cells with an elongated spindle-shape and thepresence of an oval nucleus. In some assays, cells are fixed and stainedwith Hema 3 before enumerating fibrocytes by direct counting, e.g.,using an inverted microscope. The amount of fibrocyte differentiation isinterpreted by one skilled in the art as an indication of a cell'sresponsiveness to SAP. As indicated by the examples of the disclosure, agreater suppression of fibrocyte differentiation indicates a greaterdegree of SAP responsiveness. An alternative method of measuringfibrocyte differentiation involves determining the expression offibrocyte-specific cell surface markers or secreted factors ,e.g.,cytokines (e.g., IL-1ra, ENA-78/CXCL-5, PAI-1), fibronectin, collagen-1.Methods of detecting and/or quantifying cell surface markers or secretedfactors are well known in the art, including but not limited to variousELISA- and FACS-based techniques using immunoreactive antibodies againstone or more fibrocyte specific markers. As described in the examples ofthe disclosure, measuring the expression of Macrophage Derived Chemokine(MDC) is an effective method of determining fibrocyte differentiation.

Methods for detecting and/or characterizing N-glycosylation (e.g.,altered N-glycosylation) of a SAP polypeptide include DNAsequencer-assisted (DSA), fluorophore-assisted carbohydrateelectrophoresis (FACE) or surface-enhanced laser desorption/ionizationtime-of-flight mass spectrometry (SELDI-TOF MS). For example, ananalysis can utilize DSA-FACE in which, for example, glycoproteins aredenatured followed by immobilization on, e.g., a membrane. Theglycoproteins can then be reduced with a suitable reducing agent such asdithiothreitol (DATA) or β-mercaptoethanol. The sulfhydryl groups of theproteins can be carboxylated using an acid such as iodoacetic acid.Next, the N-glycans can be released from the protein using an enzymesuch as N-glycosidase F. N-glycans, optionally, can be reconstituted andderivatized by reductive amination. The derivatized N-glycans can thenbe concentrated. Instrumentation suitable for N-glycan analysisincludes, for example, the ABI PRISM® 377 DNA sequencer (AppliedBiosystems). Data analysis can be performed using, for example,GENESCAN®. 3.1 software (Applied Biosystems). Optionally, isolatedmannoproteins can be further treated with one or more enzymes to confirmtheir N-glycan status. Exemplary enzymes include, for example,a-mannosidase or α-1,2 mannosidase,. Additional methods of N-glycananalysis include, for example, mass spectrometry (e.g., MALDI-TOF-MS),high-pressure liquid chromatography (HPLC) on normal phase, reversedphase and ion exchange chromatography (e.g., with pulsed amperometricdetection when glycans are not labeled and with UV absorbance orfluorescence if glycans are appropriately labeled). See also Callewaertet al. (2001) Glycobiology 11(4):275-281 and Freire et al. (2006)Bioconjug. Chem. 17(2):559-564, the disclosures of each of which areincorporated herein by reference in their entirety.

Treatment Methods

In one aspect, the disclosure provides methods for treating anSAP-responsive disorder in a patient by administering a therapeuticallyeffective amount of a variant SAP polypeptide of the invention to apatient in need thereof. The dosage and frequency of treatment can bedetermined by one skilled in the art and will vary depending on thesymptoms, age and body weight of the patient, and the nature andseverity of the disorder to be treated or prevented. In someembodiments, a variant SAP polypeptide is administered to a patient onceor twice per day, once or twice per week, once or twice per month, orjust prior to or at the onset of symptoms.

Dosages may be readily determined by techniques known to those of skillin the art or as taught herein. Toxicity and therapeutic efficacy of SAPmay be determined by standard pharmaceutical procedures in experimentalanimals, for example, determining the LD₅₀ and the ED₅₀. The ED₅₀(Effective Dose 50) is the amount of drug required to produce aspecified effect in 50% of an animal population. The LD₅₀ (Lethal Dose50) is the dose of drug which kills 50% of a sample population.

In some embodiments, the SAP-responsive disorder is fibrosis. The use ofSAP as a therapeutic treatment for fibrosis is described in U.S. PatentApplication No. 2007/0243163, which is hereby incorporated by reference.Fibrosis related disorders that may be amenable to treatment with thesubject method include, but are not limited to, collagen disease,interstitial lung disease, human fibrotic lung disease (e.g.,obliterative bronchiolitis, idiopathic pulmonary fibrosis, pulmonaryfibrosis from a known etiology, tumor stroma in lung disease, systemicsclerosis affecting the lungs, Hermansky-Pudlak syndrome, coal worker'spneumoconiosis, asbestosis, silicosis, chronic pulmonary hypertension,AIDS-associated pulmonary hypertension, sarcoidosis, moderate to severeasthma and the like), fibrotic vascular disease, arterial sclerosis,atherosclerosis, varicose veins, coronary infarcts, cerebral infarcts,myocardial fibrosis, musculoskeletal fibrosis, post-surgical adhesions,human kidney disease (e.g., nephritic syndrome, Alport syndrome,HIV-associated nephropathy, polycystic kidney disease, Fabry's disease,diabetic nephropathy, chronic glomerulonephritis, nephritis associatedwith systemic lupus, and the like), progressive systemic sclerosis(PSS), primary scalloping cholangitis (PSC), liver fibrosis, livercirrhosis, renal fibrosis, pulmonary fibrosis, cystic fibrosis, chronicgraft versus host disease, scleroderma (local and systemic), Grave'sophthalmopathy, diabetic retinopathy, glaucoma, Peyronie's disease,penis fibrosis, urethrostenosis after cystoscope, inner accretion aftersurgery, scarring, myelofibrosis, idiopathic retroperitoneal fibrosis,peritoneal fibrosis from a known etiology, drug-induced ergotism,fibrosis incident to benign or malignant cancer, fibrosis incident tomicrobial infection (e.g., viral, bacterial, parasitic, fungal, etc.),Alzheimer's disease, fibrosis incident to inflammatory bowel disease(including stricture formation in Crohn's disease and microscopiccolitis), stromal cell tumors, mucositis, fibrosis induced by chemicalor environmental insult (e.g., cancer chemotherapy, pesticides, orradiation (e.g., cancer radiotherapy)).

In some embodiments, the fibrosis related disorder is selected fromsystemic or local scleroderma, keloids, hypertrophic scars,atherosclerosis, restenosis, pulmonary inflammation and fibrosis,idiopathic pulmonary fibrosis, liver cirrhosis, fibrosis as a result ofchronic hepatitis B or C infection, kidney disease, heart diseaseresulting from scar tissue, macular degeneration, and retinal andvitreal retinopathy. In some embodiments, the fibrosis related disorderresults from chemotherapeutic drugs, radiation-induced fibrosis, andinjuries and burns. In some embodiments, the fibrosis-related disorderor condition results from post-surgical scarring, e.g., followingtrabeculectomy or other filtration surgery of the eye.

In some embodiments, the SAP-responsive disorder is a hypersensitivitydisorder such as those mediated by Th1 or Th2 responses. The use of SAPas a therapeutic treatment for hypersensitivity disorders is alsodescribed in U.S. Provisional Application No. 61/209,795, which ishereby incorporated by reference. Hypersensitivity related disordersthat may be amenable to treatment with SAP include, but are not limitedto, allergic rhinitis, allergic sinusitis, allergic conjunctivitis,allergic bronchoconstriction, allergic dyspnea, allergic increase inmucus production in the lungs, atopic eczema, dermatitis, urticaria,anaphylaxis, pneumonitis, and allergic-asthma.

In some embodiments, a variant SAP polypeptide of the invention may beused to treat allergen-specific immune responses, such as anaphylaxis,to various antigens, including, but not limited to, antimicrobials(e.g., cephalosporins, sulfonamides, penicillin and other β-lactams),anticonvulsants (e.g., phenytoin, phenobarbital, carbamazepine, dapsone,allopurinol, and minocycline), chemotherapeutics (e.g., taxanes,platinum compounds, asparaginases, and epipodophyllotoxins), heparin,insulin, protamine, aspirin and other non-steroidal anti-inflammatorydrugs, muscle relaxants (e.g., succinylcholine, atracurium, vecuronium,and pancuronium), induction agents (e.g., barbiturates, etomidate,propofol), narcotics (e.g., fentanyl, meperidine, morphine), colloidsfor intravascular volume expansion, radiocontrast materials, bloodproducts, latex, animal products, animal dander, dust mites, insects(e.g., bites, stings or venom), cosmetics, metals (e.g., nickel, cobalt,and chromate), plants, spores, pollen, food (e.g., milk, eggs, wheat,soy, peanuts and tree nuts, seafood), vaccination, and fungal antigens(e.g., Aspergillus, Curvularia, Exserohilum, and Alternaria species).

In some embodiments, the SAP-responsive disorder is an autoimmunedisorder such as those mediated by Th1 or Th2 responses. The use of SAPas a therapeutic treatment for autoimmune disorders is also described inU.S. Provisional Application No 61/209,845, which is hereby incorporatedby reference. Autoimmune related disorders that may be amenable totreatment with SAP include, but are not limited to, type I diabetes,multiple sclerosis, rheumatoid arthritis, psoriatic arthritis,autoimmune myocarditis, pemphigus, myasthenia gravis, Hashimoto'sthyroiditis, Graves' disease, Addison's disease, autoimmune hepatitis,chronic Lyme arthritis, familial dilated cardiomyopathy, juveniledermatomyositis, polychondritis, Sjogren's syndrome, psoriasis, juvenileidiopathic arthritis, inflammatory bowel disease, systemic lupuserythematosus, chronic obstructive pulmonary disease, andgraft-versus-host disease.

In some embodiments, the SAP-responsive disorder is a mucositis. The useof SAP as a therapeutic treatment for mucositis is also described inU.S. application Ser. No. 12/217,614, which is hereby incorporated byreference. Methods of the invention may be useful for treating oral,esophageal, and gastrointestinal mucositis, as well as gastric andduodenal ulcers, or erosions of the stomach and esophagus.

In some embodiments, a variant SAP polypeptide of the invention may beused to treat an inflammatory disease or condition. In some embodiments,the inflammatory disease may be a viral, bacterial, fungal, or parasiticinfection. The use of SAP as a therapeutic treatment for viral infectionhas also been described in U.S. Pat. No. 6,406,698 and in InternationalPatent Application No. WO1997/026906, which are both incorporated byreference herein.

Pharmaceutical Preparations and Formulations

In certain embodiments, the methods described herein involveadministration of at least one variant SAP polypeptide of the inventionto a subject as a therapeutic agent. The therapeutic agents of theinvention may be formulated in a conventional manner using one or morephysiologically acceptable carriers or excipients. For example,therapeutic agents and their physiologically acceptable salts andsolvates may be formulated for administration by, for example, injection(e.g. SubQ, IM, IP), inhalation or insufflation (either through themouth or the nose) or oral, buccal, sublingual, transdermal, nasal,parenteral or rectal administration. In certain embodiments, therapeuticagents may be administered locally, at the site where the target cellsare present, i.e., in a specific tissue, organ, or fluid (e.g., blood,cerebrospinal fluid, tumor mass, etc.).

The present invention further provides use of any variant SAPpolypeptide of the invention in the manufacture of a medicament for thetreatment or prevention of a disorder or a condition, as describedherein, in a patient, for example, the use of a variant SAP polypeptidein the manufacture of medicament for the treatment of a disorder orcondition described herein. In some aspects, any variant SAP polypeptideof the invention may be used to make a pharmaceutical preparation forthe use in treating or preventing a disease or condition describedherein.

Therapeutic agents can be formulated for a variety of modes ofadministration, including systemic and topical or localizedadministration. Techniques and formulations generally may be found inRemington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA.For parenteral administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the compounds can be formulated in liquid solutions,preferably in physiologically compatible buffers such as Hank's solutionor Ringer's solution. In addition, the compounds may be formulated insolid form and dissolved or suspended immediately prior to use.Lyophilized forms are also included. In some embodiments, thetherapeutic agents can be administered to cells by a variety of methodsknow to those familiar in the art, including, but not restricted to,encapsulation in liposomes, by iontophoresis, or by incorporation intoother vehicles, such as hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesive microspheres.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets, lozenges, or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well known in theart. Liquid preparations for oral administration may take the form of,for example, solutions, syrups or suspensions, or they may be presentedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives such as suspendingagents (e.g., sorbitol syrup, cellulose derivatives or hydrogenatededible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

For administration by inhalation (e.g., pulmonary delivery), therapeuticagents may be conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer, with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin, for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

In the methods of the invention, the pharmaceutical compounds can alsobe administered by intranasal or intrabronchial routes includinginsufflation, powders, and aerosol formulations (for examples of steroidinhalants, see Rohatagi (1995) J. Clin. Pharmacology. 35:1187-1193; Tjwa(1995) Ann. Allergy Asthma Immunol. 75:107-111). For example, aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They alsomay be formulated as pharmaceuticals for non-pressured preparations suchas in a nebulizer or an atomizer. Typically, such administration is inan aqueous pharmacologically acceptable buffer.

Pharmaceutical compositions suitable for respiratory delivery (e.g.,intranasal, inhalation, etc.) of variant SAP polypeptides may beprepared in either solid or liquid form.

SAP polypeptides of the invention may be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

In addition, SAP polypeptides of the invention may also be formulated asa depot preparation. Such long-acting formulations may be administeredby implantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, therapeutic agents may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.Controlled release formula also includes patches.

In certain embodiments, the compounds described herein can be formulatedfor delivery to the central nervous system (CNS) (reviewed in Begley,Pharmacology & Therapeutics 104: 29-45 (2004)). Conventional approachesfor drug delivery to the CNS include: neurosurgical strategies (e.g.,intracerebral injection or intracerebroventricular infusion); molecularmanipulation of the agent (e.g., production of a chimeric fusion proteinthat comprises a transport peptide that has an affinity for anendothelial cell surface molecule in combination with an agent that isitself incapable of crossing the blood-brain-barrier in an attempt toexploit one of the endogenous transport pathways of theblood-brain-barrier); pharmacological strategies designed to increasethe lipid solubility of an agent (e.g., conjugation of water-solubleagents to lipid or cholesterol carriers); and the transitory disruptionof the integrity of the BBB by hyperosmotic disruption (resulting fromthe infusion of a mannitol solution into the carotid artery or the useof a biologically active agent such as an angiotensin peptide).

In certain embodiments, SAP polypeptides of the invention areincorporated into a topical formulation containing a topical carrierthat is generally suited to topical drug administration and comprisingany such material known in the art. The topical carrier may be selectedso as to provide the composition in the desired form, e.g., as anointment, lotion, cream, microemulsion, gel, oil, solution, or the like,and may be comprised of a material of either naturally occurring orsynthetic origin. It is preferable that the selected carrier notadversely affect the active agent or other components of the topicalformulation. Examples of suitable topical carriers for use hereininclude water, alcohols and other nontoxic organic solvents, glycerin,mineral oil, silicone, petroleum jelly, lanolin, fatty acids, vegetableoils, parabens, waxes, and the like.

Pharmaceutical compositions (including cosmetic preparations) maycomprise from about 0.00001 to 100% such as from 0.001 to 10% or from0.1% to 5% by weight of one or more of the variant SAP polypeptidesdescribed herein. In certain topical formulations, the active agent ispresent in an amount in the range of approximately 0.25 wt. % to 75 wt.% of the formulation, preferably in the range of approximately 0.25 wt.% to 30 wt. % of the formulation, more preferably in the range ofapproximately 0.5 wt. % to 15 wt. % of the formulation, and mostpreferably in the range of approximately 1.0 wt. % to 10 wt. % of theformulation.

Conditions of the eye can be treated or prevented by, e.g., systemic,topical, intraocular injection of therapeutic agents, or by insertion ofa sustained release device that releases therapeutic agents. SAPpolypeptides of the invention may be delivered in a pharmaceuticallyacceptable ophthalmic vehicle, such that the compound is maintained incontact with the ocular surface for a sufficient time period to allowthe compound to penetrate the corneal and internal regions of the eye,as for example the anterior chamber, conjunctiva, posterior chamber,vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary,lens, choroid/retina and sclera. The pharmaceutically acceptableophthalmic vehicle may, for example, be an ointment, vegetable oil or anencapsulating material. Alternatively, the compounds may be injecteddirectly into the vitreous and aqueous humour. In a further alternative,the compounds may be administered systemically, such as by intravenousinfusion or injection, for treatment of the eye.

Therapeutic agents described herein may be stored in oxygen-freeenvironment according to methods in the art.

Exemplary compositions comprise an SAP polypeptide with one or morepharmaceutically acceptable carriers and, optionally, other therapeuticingredients. The carrier(s) must be “pharmaceutically acceptable” in thesense of being compatible with the other ingredients of the compositionand not eliciting an unacceptable deleterious effect in the subject.Such carriers are described herein or are otherwise well known to thoseskilled in the art of pharmacology. In some embodiments, thepharmaceutical compositions are pyrogen-free and are suitable foradministration to a human patient. In some embodiments, thepharmaceutical compositions are irritant-free and are suitable foradministration to a human patient. In some embodiments, thepharmaceutical compositions are allergen-free and are suitable foradministration to a human patient. The compositions may be prepared byany of the methods well known in the art of pharmacy.

In some embodiments, an SAP polypeptide is administered in a timerelease formulation, for example in a composition which includes a slowrelease polymer. An SAP polypeptide can be prepared with carriers thatwill protect against rapid release. Examples include a controlledrelease vehicle, such as a polymer, microencapsulated delivery system,or bioadhesive gel. Alternatively, prolonged delivery of an SAPpolypeptide may be achieved by including in the composition agents thatdelay absorption, for example, aluminum monostearate hydrogels andgelatin.

Methods for delivering nucleic acid compounds are known in the art (see,e.g., Akhtar et al., 1992, Trends Cell Bio., 2, 139; and DeliveryStrategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995;Sullivan et al., International Application No. WO 94/02595). Theseprotocols can be utilized for the delivery of virtually any nucleic acidcompound. Nucleic acid compounds can be administered to cells by avariety of methods known to those familiar to the art, including, butnot restricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres. Alternatively,the nucleic acid/vehicle combination is locally delivered by directinjection or by use of an infusion pump. Other routes of deliveryinclude, but are not limited to, oral (tablet or pill form) and/orintrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1158). Otherapproaches include the use of various transport and carrier systems, forexample though the use of conjugates and biodegradable polymers. For acomprehensive review on drug delivery strategies, see Ho et al., 1999,Curr. Opin. Mol. Ther., 1, 336-343 and Jain, Drug Delivery Systems:Technologies and Commercial Opportunities, Decision Resources, 1998 andGroothuis et al., 1997, J. NeuroVirol., 3, 387-400. More detaileddescriptions of nucleic acid delivery and administration are provided inSullivan et al., supra, Draper et al., PCT WO93/23569, Beigelman et al.,International Application No. WO99/05094, and Klimuk et al.,International Publication No. WO99/04819.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.

EXEMPLIFICATION EXAMPLE 1: RECOMBINANT SAP IS MORE POTENT THAN HUMANSERUM-DERIVED SAP

Recombinant human SAP isolated from CHO-S cells (rhSAP) and humanserum-derived SAP (hSAP) were assayed for bioactivity using an in vitrobioassay. In this assay, monocyte enriched Peripheral Blood MononuclearCells (PBMCs) were incubated with varying concentrations of either rhSAPor hSAP for 96 hours. Following this incubation, resulting culturesupernatants were removed and assayed by ELISA to quantify the amount ofMacrophage Derived Chemokine (MDC) that was produced. MDC is produced byfibrocytes and therefore an indicator of monocyte differentiation intofibrocytes. By comparing the inhibitory concentration, 50% (IC₅₀) of thesample to the hSAP reference standard, the relative potency of a SAPglycovariant can be determined. The result is expressed as an IC₅₀ ratioof the sample versus the hSAP reference standard.

All SAP samples and standards were initially diluted to a concentrationof 1.0 mg/mL in Supplemented FibroLife Media. SAP standards wereserially diluted to generate working standard concentrations of 60, 30,20, 13.4, 8.8, 6.0, 3.0, 1.5, and 0.75 μg/mL (final standardconcentration of 30, 15, 10, 6.7, 4.4, 3.0. 1.5, 0.75, and 0.375 μg/mL).See the following Table 1.

Working rhSAP Standard Concentration Volume of Supplemented (μg/mL)Volume of Standard FibroLife Media 60 60 (1 mg/mL) 940 30 600 (60 μg/mL)600 20 800 (30 μg/mL Std) 400 13.4 800 (20 μg/mL Std) 400 8.8 800 (13.4μg/mL Std) 400 6.0 800 (8.8 μg/mL Std) 400 3.0 600 (6.0 μg/mL Std) 6001.5 600 (3.0 μg/mL Std) 600 0.75 600 (1.5 μg/mL Std) 600

To prepare for the ELISA assay, the capture antibody (i.e., mouseanti-human MDC) was diluted to the working concentration in PBS withoutcarrier protein. The diluted capture antibody was used to coat 96-wellplates, and then each plate was sealed and incubated overnight at roomtemperature. Before using the coated plates, each well was aspirated andwashed with wash buffer, repeating the process two times for a total ofthree washes. The plates were then blocked by adding 300 μL of reagentdiluent to each well and incubating at room temperature for one hour.After incubation the aspiration and well-washing procedure was repeated.

For the ELISA assay, 100 μL samples of either the supernatants from themonocyte/fibrocyte cultures or the SAP standards were added to eachwell. The plate was then incubated at room temperature for 2 hoursbefore aspirating and washing the wells. Then 100 μL of a workingdilution of Streptavidin-HRP was added to each well. The plate wasincubated for 20 minutes at room temperature before adding 50 μL of StopSolution to each well. Immediately, the optical density of each well wasmeasured using a microplate reader set to 450 nm. If wavelengthcorrection was available, the microplate reader was set to 540 nm or 570nm. If wavelength correction was not available, then the readings at 540nm or 570 nm were subtracted from the readings at 450 nm. Thissubtraction corrects for optical imperfections in the plate.

Both RHSAP and hSAP were assayed for bioactivity using this assay (FIG.1 ). On average, RHSAP is 3.4-fold more active than hSAP (average of 7independent experiments).

EXAMPLE 2: MODIFICATION OF SAP GLTYCAN STRUCTURES

Using in vitro glycoremodeling techniques, glycan moieties on a sampleof recombinant human SAP (rhSAP) were modified to replace terminalα2,3-linked sialic acid moieties with α2,6-linked sialic acid moieties(FIG. 2 , A and B). Similarly, a sample of human serum-derived SAP(hSAP) was also modified to replace terminal α2,6-linked sialic acidmoieties with α2,3-linked sialic acid moieties (FIG. 2 , C and D). Inaddition, as rhSAP isolated from CHO-S cells is only partiallysialylated, a sample of rhSAP was treated to fully sialylate theattached glycan structures with α2,3-linked sialic acid moieties (FIG. 2, E and F).

Both rhSAP and hSAP (Calbiochem Cat#970549) were treated with aα2,3,6,8,9-sialidase (Sigma Cat #N8271) to fully desialylate thepolypeptides. After sialidase treatment for 17 hours, desialylated (i.e.asialo) rhSAP and hSAP were purified using phosphoethanolamine (PE)affinity and size exclusion (Sephadex 200 prep grade) chromatography.Purified asialo SAP polypeptides were then enzymatically treated usingeither α2,3- or α2,6-sialyltransferases (Calbiochem Cat #566223) in thepresence of CMP-sialic acid (Calbiochem Cat #233264) for 17 hours at 37°C. The following tables provide details on each reaction mixture.

Asialo rhSAP Reactions (Rxns) Treatment with α2,3 sialyltransferase(ST3Gal3) parameter units value rhSAP, ST3Gal3 [asialo rhSAP] stk mg/mL10.8 reagent stocks asialo rhSAP MW g/mol 116293 [asialo rhSAP] stk μM93 max [Galactose] μM 929 [ST3Gal3] stk mg/mL 0.9 ST3Gal3 MW g/mol 84000[ST3Gal3] stk μM 10.7 [CMP-SA] stk mg/mL 25 CMP-SA MW g/mol 658.4[CMP-SA] stk mM 38 rxn volumes μL asialo rhSAP in rxn μL 260 μL ST3Gal3in rxn μL 50 μL CMP-SA in rxn μL 75 buffer (10 mM HEPES/ μL 615 150 mMNaCl, pH 8) tot rxn vol μL 1000 rxn [asialo rhSAP] rxn μM 24.1concentrations max [Galactose] μM 241 [SAP] rxn mg/mL 2.8 [ST3Gal3] rxnμM 0.5357 [ST3Gal3] rxn mg/mL 0.045 [CMP-SA] rxn mM 2.85 asialorhSAP:ST3 mass 62 ratio asialo rhSAP:ST3 molar 45 ratio CMP-SA:ST3 molar5316 ratio CMP-SA:asialo rhSAP molar 118 ratio CMP-SA:Galactose molar11.8 ratio

Treatment with α2,6 sialyltransferase (ST6 Rxn) parameter units valuerhSAP, ST6 [asialo rhSAP] stk mg/mL 10.8 reagent stocks asialo rhSAP MWg/mol 116293 [asialo rhSAP] stk μM 93 max [Galactose] μM 929 [ST6] stkmg/mL 0.205 ST6 MW g/mol 42000 [ST6] stk μM 4.9 [CMP-SA] stk mg/mL 25CMP-SA MW g/mol 658.4 [CMP-SA] stk mM 38 rxn volumes μL asialo rhSAP 1in rxn μL 260 μL ST6 in rxn μL 30 μL CMP-SA in rxn μL 75 buffer (10 mMHEPES/ μL 635 150 mM NaCl, pH 8) tot rxn vol μL 1000 rxn [asialo rhSAP]rxn μM 24.1 concentrations max [Galactose] μM 241 [asialo rhSAP] rxnmg/mL 2.8 [ST6] rxn μM 0.146 [ST6] rxn mg/mL 0.006 [CMP-SA] rxn mM 2.85asialo rhSAP:ST6 mass 457 ratio asialo rhSAP:ST6 molar 165 ratioCMP-SA:ST6 molar 19448 ratio CMP-SA:asialo rhSAP molar 118 ratioCMP-SA:Galactose molar 11.8 ratio

Asialo hSAP Rxns Treatment with α2,3 sialyltransferase (ST3Gal3)parameter units value hSAP, ST3Gal3 [asialo hSAP] stk mg/mL 5.6 reagentstocks SAP MW g/mol 116293 [asialo hSAP] stk μM 48 max [Galactose] μM482 [ST3Gal3] stk mg/mL 0.9 ST3Gal3 MW g/mol 84000 [ST3Gal3] stk μM 10.7[CMP-SA] stk mg/mL 25 CMP-SA MW g/mol 658.4 [CMP-SA] stk mM 38 rxnvolumes μL asialo hSAP in rxn μL 500 μL ST3Gal3 in rxn μL 50 μL CMP-SAin rxn μL 75 buffer (10 mM HEPES/ μL 375 150 mM NaCl, pH 8) tot rxn volμL 1000 rxn [asialo hSAP] rxn μM 24.1 concentrations max [Galactose] μM241 [asialo hSAP] rxn mg/mL 2.8 [ST3Gal3] rxn μM 0.54 [ST3Gal3] rxnmg/mL 0.045 [CMP-SA] rxn mM 2.85 asialo hSAP:ST3 mass 62 ratio asialohSAP:ST3 molar 45 ratio CMP-SA:ST3 molar 5316 ratio CMP-SA:asialo hSAPmolar 118 ratio CMP-SA:Galactose molar 11.8 ratio

Treatment with α2,6 sialyltransferase (ST6 Rxn) parameter units valuehSAP, ST6 [asialo hSAP] stk mg/mL 5.6 reagent stocks asialo hSAP MWg/mol 116293 [asialo hSAP] stk μM 48 max [Galactose] μM 482 [ST6] stkmg/mL 0.205 ST6 MW g/mol 42000 [ST6] stk μM 4.9 [CMP-SA] stk mg/mL 25CMP-SA MW g/mol 658.4 [CMP-SA] stk mM 38 rxn volumes μL asialo hSAP inrxn μL 500 μL ST6 in rxn μL 30 μL CMP-SA in rxn μL 75 buffer (10 mMHEPES/ μL 395 150 mM NaCl, pH 8) tot rxn vol μL 1000 rxn [asialo hSAP]rxn μM 24.1 concentrations max [Galactose] μM 241 [asialo hSAP] rxnmg/mL 2.8 [ST6] rxn μM 0.146 [ST6] rxn mg/mL 0.006 [CMP-SA] rxn mM 2.85asialo hSAP:ST6 mass 455 ratio asialo hSAP:ST6 molar 164 ratioCMP-SA:ST6 molar 19448 ratio CMP-SA:asialo hSAP molar 118 ratioCMP-SA:Galactose molar 11.8 ratio

In a separate reaction, complete sialylation of rhSAP using theα2,3-sialyltransferase without first desialylating the molecule waspreformed at 37° C. for 17 hours according to the following table.

Treatment with α2,3 sialyltransferase (ST3Gal3) parameter units valuerhSAP ST3Gal3 [rhSAP] stk mg/mL 19.0 reagent stocks rhSAP MW g/mol116293 [rhSAP] stk μM 163 max [Galactose] μM 1634 [ST3Gal3] stk mg/mL0.9 ST3Gal3 MW g/mol 84000 [ST3Gal3] stk μM 10.7 [CMP-SA] stk mg/mL 25CMP-SA MW g/mol 658.4 [CMP-SA] stk mM 38 rxn volumes μL rhSAP in rxn μL500 μL ST3Gal3 in rxn μL 50 μL CMP-SA in rxn μL 100 buffer (10 mM HEPES/μL 350 150 mM NaCl, pH 8) tot rxn vol μL 1000 rxn [rhSAP] rxn μM 81.7concentrations max [Galactose] μM 817 [rhSAP] rxn mg/mL 9.5 [ST3Gal3]rxn μM 0.54 [ST3Gal3] rxn mg/mL 0.045 [CMP-SA] rxn mM 3.80 BTA-02-17:ST3mass 211 ratio BTA-02-17:ST3 molar 152 ratio CMP-SA:ST3 molar 7088 ratioCMP-SA:BTA-02-17 molar 46 ratio CMP-SA:Galactose molar 4.6 ratio

After sialylation treatment, both sialylated rhSAP and hSAP variantswere purified using PE affinity chromatography followed by dialysis into10 mM NaPi/5% sorbitol pH 7.5 (P5S buffer). Confirmation of the desiredsialic acid linkages (i.e, α2,3-linked hSAP and α2,6-linked RHSAP) waspreformed using both Liquid Chromatography Mass Spectrometry (FIG. 2 ;A, C, and E) and Anion-Exchange High Performance Liquid Chromatography(FIG. 2 ; B, D, and F) following treatment of the glycovariant SAPpolypeptides with α2,3 sialidase (Calbiochem Cat#480706) at 37° C. for24 hours.

EXAMPLE3: IN VITRO BIOASSAYS TO DETERMINE SAP GLYCOVARIANT POTENCY FORINHIBITING MONOCYTE DIFFERENTIATION INTO FIBROCYTES

Glycoremodeled rhSAP and hSAP were assayed for bioactivity using thesame in vitro bioassay described in Example 1. In brief, monocyteenriched peripheral blood mononuclear cells (PBMCs) were incubated withvarying concentrations of an SAP polypeptide for 96 hours. Followingthis incubation, resulting culture supernatants were removed and assayedby ELISA to quantify the about of Macrophage Derived Chemokine (MDC)that was produced. The results were expressed as an IC₅₀ ratio of thesample versus the hSAP reference standard and plotted as relativeactivity (relative activity of hSAP=1). All α2,3-sialic acid linkagecontaining test materials are ≥2.4-fold more active than hSAP (FIG. 3 ).Equal mixtures of α2,3-and α2,6-linked sialic acid derivatives of SAPshow intermediate activity levels between 100% α2,6-linked and 100%α2,3-linked SAP as expected (two rightmost bars in FIG. 3 ).

An alternative method of quantifying fibrocyte differentiation involvesdirectly enumerating the number of fibrocytes that are generated aftermonocytes are incubated with a fibrocyte suppressant (e.g., SAP orvariant thereof) or activating agent (e.g., M-CSF). In one experiment,monocytes were purified from whole blood-derived PBMC using negativemagnetic bead selection standard in the art (e.g. CAT# 113-41D,Invitrogen, Carlsbad, Calif.) and cultured in a 96-Well tissue cultureplate containing FibroLife Media supplemented with 25 or 50 ng/ml ofM-CSF in triplicate. The plate was incubated for 96 hours at 37 ° C. ina 5% CO2 incubator. The cells were then fixed with paraformaldehyde andstained with Hema 3 stain (Cat # 122-911, Hema 3 Stain, FisherScientific, Hampton, N.H.). The number of fibrocytes per well weredetermined by summing the count of five different fields per well usingan inverted microscope. Fibrocytes were defined morphologically asadherent cells with an elongated spindle-shape and the presence of anoval nucleus. The data indicated that either 25 or 50 ng/ml of M-CSF wassufficient to increase the number of fibrocytes differentiating frommonocytes by ˜50% in this donor (FIG. 4 ). Subsequent experiments usedFibroLife Media supplemented with 25 ng/ml of M-CSF as needed anddefined below.

-   -   Fibrolife Media: (Cat # LM-0001, Lifeline Cell Technology,        Walkersville, Md.) supplemented with 10 mM HEPES (Cat # H0887,        Sigma-Aldrich), 1×non-essential amino acids (Cat # M7145,        Sigma-Aldrich,), 1 mM sodium pyruvate (Cat # S8636,        Sigma-Aldrich), 2 mM glutamine (Cat # 25030-149, Invitrogen),        100 U/ml penicillin and100 ug/ml streptomycin (Cat # P0781,        Sigma-Aldrich), and ITS-3 (Cat # 12771, 500 ug/ml bovine serum        albumin, 10 ug/ml insulin, 5 □ug/ml transferrin, 5 ng/ml sodium        selenite, 5 ug/ml linoleic acid, and 5 ug/ml oleic acid;        Sigma-Aldrich).

In an additional experiment, PBMC or monocytes were purified from wholeblood and cultured in FibroLife Media supplemented with various amountsof SAP in triplicate (as described above). The plate was incubated for96 hours at 37° C. in a 5% CO2 incubator. The cells were then fixed withparaformaldehyde and stained with Hema 3 stain (Cat # 122-911, Hema 3Stain, Fisher Scientific, Hampton, N.H.). The number of fibrocytes perwell were determined by summing the count of five different fields perwell using an inverted microscope. The minimum concentration of SAPnecessary to provide maximum inhibition of fibrocyte differentiation inthis system was determined to be 2ug/m1 (FIG. 5 ). The number offibrocytes decreases with increasing SAP concentration in all donors.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will be apparent to those skilled in the art upon review ofthis specification and the below-listed claims. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. A glycosylated human Serum Amyloid P (SAP) polypeptide comprising anN-linked oligosaccharide chain, wherein at least one branch of theoligosaccharide chain terminates with a α2,3-linked sialic acid moiety.2. (canceled)
 3. The glycosylated human SAP polypeptide of claim 1,wherein all branches of the oligosaccharide chain terminate withα2,3-linked sialic acid moieties.
 4. The glycosylated human SAPpolypeptide of claim 1, wherein the oligosaccharide chain issubstantially free of α2,6-linked sialic acid moieties.
 5. Theglycosylated human SAP polypeptide of claim 1, wherein the polypeptidecomprises an amino acid sequence at least 95% identical to SEQ ID NO: 1.6. The glycosylated human SAP polypeptide of claim 1, wherein thepolypeptide is a fusion protein comprising an SAP domain and one or moreheterologous domains.
 7. The glycosylated human SAP polypeptide of claim1, wherein the polypeptide comprises one or more modified amino acidresidues.
 8. The glycosylated human SAP polypeptide of claim 7, whereinthe one or more modified amino acid residues comprise a PEGylated aminoacid, a prenylated amino acid, an acetylated amino acid, a biotinylatedamino acid, and/or an amino acid conjugated to an organic derivatizingagent.
 9. The glycosylated human SAP polypeptide of claim 1, wherein theSAP polypeptide has an IC50 for inhibiting the differentiation ofmonocytes into fibrocytes in vitro that is less than one-half that of acorresponding sample of wild-type SAP isolated from human serum.
 10. Apharmaceutical preparation suitable for use in a mammal comprising thehuman SAP polypeptide of claim 1 and a pharmaceutically acceptablecarrier.
 11. A method of treating or preventing a-disorder or conditionin a patient, the method comprising administering to a patient in needthereof a therapeutically effective amount of the SAP polypeptide ofclaim 1, wherein the disorder or condition is selected from a fibroticor fibroproliferative disorder or condition, a hypersensitivity disorderor condition, an autoimmune disorder or condition, an inflammatorydisorder or condition, and mucositis.
 12. A method of making a human SAPpolypeptide, comprising: i) expressing a human SAP polypeptide in a CHOcell; and ii) isolating the human SAP polypeptide from the cell.
 13. Themethod of claim 12, wherein the isolated SAP polypeptide comprises anN-linked oligosaccharide chain, and wherein at least one branch of theoligosaccharide chain terminates with a α2,3-linked sialic acid moiety.14. The method claim 12, wherein the isolated SAP polypeptide comprisesan N-linked oligosaccharide chain, and wherein the oligosaccharide chainhas at least 50% fewer α2,6-linked sialic acid moieties than wild-typeSAP isolated from human serum.
 15. The method of claim 12, wherein theisolated human SAP polypeptide has an IC₅₀ for inhibiting thedifferentiation of monocytes into fibrocytes in vitro that is less thanone-half that of a corresponding sample wild-type SAP isolated fromhuman serum.
 16. The method of claim 12, further comprisingenzymatically or chemically altering the isolated SAP polypeptide toproduce an SAP polypeptide having a modified oligosaccharide chain. 17.The method of claim 16, wherein the process of enzymatically orchemically altering the isolated SAP polypeptide removes one or moreterminal α2,6-linked sialic acid moieties from the oligosaccharidechain.
 18. The method of claim 16, wherein the process of enzymaticallyor chemically altering the isolated SAP polypeptide replaces one or moreterminal α2,6-linked sialic acid moieties on the oligosaccharide chainwith one or more α2,3-linked sialic acid moieties. 19-26. (canceled)